Use of attenuated strains of parasites for the prevention or treatment of pathologies associated with an apicomplexan

ABSTRACT

Strains of Sarcocystidae selected from  Toxoplasma  spp or  Neospora  spp isolated from their natural environment and having an immunostimulant effect, for the use thereof in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae.

The present invention relates to the use of attenuated strains ofparasites for preventing or treating pathologies associated with anapicomplexan.

The Apicomplexa are predominantly obligate intracellular parasites thathave a life cycle that may involve several hosts. The phylum of theseparasites is subdivided into several families.

Toxoplasma gondii (T. gondii) belongs to the Sarcocystidae family. Thisprotozoon exists in three infectious forms which vary depending on thehost and the infectious stage:

-   -   tachyzoite: the infectious proliferative form which multiplies        asexually in the cells of the intermediate hosts (i.e. all        homeotherms) and definitive hosts (i.e. felids and the cat in        particular),    -   bradyzoite: slowly dividing form with low level of metabolism of        the parasite contained in cysts,    -   sporozoite: the form contained in the oocysts, which results        from the sexual multiplication of the parasite in the intestine        of the definitive hosts (i.e. cat and other felids).        The cat, the definitive host of the parasite, becomes infected        by ingesting parasitized prey containing cysts (or tachyzoites        if the animal is in the acute phase of toxoplasmosis), or by        ingestion of oocysts. After sexual multiplication in the        intestine of the felids, via gametocytes, the oocysts are        disseminated in the environment. These oocysts sporulate in the        external environment and they remain pathogenic for at least a        year. After ingestion of oocysts, the released sporozoites        infect the enterocytes of the definitive or intermediate host        and undergo transformation to tachyzoites, which are        disseminated in the organism. Under the pressure of the immune        system, the tachyzoites become encysted with preferential        tropism for the central nervous system, the retina or the        muscles. Ingestion of encysted tissues is the second commonest        cause of contamination of the definitive and intermediate hosts.        After ingestion of cysts, the bradyzoites are released and        infect the intestinal epithelial cells and are transformed into        tachyzoites, which are disseminated in the host.

The strains of T. gondii are classified according to their degree ofvirulence in vivo: the type I strains (i.e. the strain RH) are highlyvirulent whereas the type II strains (i.e. the strains ME49, 76K or Pru)and type III strains (i.e. the strains CEP or M7741) are less virulentand generally establish chronic infections. Moreover, numerous atypicalstrains not assignable to the first three types have been identified, inparticular in Africa and in South America (Howe D K et al., 1995, J.Infect. Dis., 171, 1561-1566; Rajendran C et al., 2011, Infect. Genet.Evol., 12, 359-368; Mercier A et al., 2010, PLoS Negl. Trop. Dis., 4,e876).

Recently, an attenuated live strain of Toxoplasma gondii, the parasiteresponsible for toxoplasmosis, was developed by silencing two genescoding for the proteins TgMIC1 and TgMIC3 (EP 1 703 914 B1 and U.S. Pat.No. 7,964,185 B2/Cérède et al., 2005, J. Exp. Med., 201: 453-63). Thisstrain, designated Toxo mic1-3 KO, generated a strong and specificimmune response against Toxoplasma gondii and makes it possible toprevent the effects of subsequent infection in the mouse (Ismaël et al.,2005, J. Infect. Dis., 194: 1176-1183) and in the ewe (Mevelec et al.,2010, Vet. Res., 41: 49). It has also been demonstrated that virulencein vivo is only very slightly affected by isolated inactivation ofTgMIC1 or of TgMIC3; in contrast, it is greatly reduced by simultaneousinactivation of both proteins, demonstrating the synergistic role of thetwo proteins (Cérède et al., 2005 J. Exp. Med., 201: 453-63).

Neospora caninum is an intracellular parasite, responsible forneosporosis. It also belongs to the Sarcocystidae family. The life cycleof Neospora caninum is very similar to that of T. gondii with twodistinct phases: a sexual phase in the final host (i.e. the canids andthe dog in particular) which leads to the production of oocysts,containing sporozoites, which are eliminated in the faeces, and anasexual phase in an intermediate host (i.e. ovines, caprins, bovines,equines, etc.), which leads to the production of tachyzoites and thencysts containing the bradyzoites.

More recently, an attenuated live strain of Neospora caninum wasobtained, the strain Neo ncmic1-3 KO, in which the ncmic1 and ncmic3genes were knocked out by homologous recombination. In this strain, thencmic3 gene is replaced by a DHFR cassette, which confers resistance topyrimethamine, and the ncmic1 gene is replaced by a CAT-GFP cassette,which endows the parasite with resistance to chloramphenicol and makesthe parasite fluorescent. The parasite no longer expresses the NcMIC1and NcMIC3 proteins. It has been shown that this mutant strain hasinfectious and immunogenic properties, endowing mammals with vaccineprotection against the harmful effects of neosporosis.

Toxoplasma gondii and Neospora caninum have in common a specific processof invasion of the host cells in several steps leading to the formationof a parasitophorous vacuole in which the parasite multiplies anddevelops.

The Cryptosporidiidae constitute another family belonging to the phylumof the apicomplexans and are responsible for cryptosporidiosis, anextremely common disease which in particular affects humans and manyanimal species including farm animals (ovin, caprin, bovine, etc.) bythe ingestion of parasites present in their food. The parasite thenmultiplies in the intestines, firstly asexually and then, secondly,sexually. The contaminated individuals excrete and disseminate newparasites, thus contaminating their environment (i.e. pastureland,water, etc.).

Several species of Cryptosporidiidae have been identified, among whichCryptosporidium parvum is one of the commonest and most virulent.

In humans, cryptosporidiosis is generally a benign disease. However, theconsequences of this disease, the incidence of which is increasing everyyear, in particular in the United States, may be extremely serious inyoung children and immunodepressed persons, especially patients infectedwith the HIV virus. Thus, within these populations, cryptosporidiosis isresponsible for severe diarrhoea that may cause significant dehydrationor, without suitable treatment, even death of the individual.

In immunocompetent adult animals, cryptosporidiosis is also benign.Conversely, cryptosporidiosis generally has grave consequences in veryyoung animals, a few days to a few weeks old, and whose immune system isimmature. Thus, Cryptosporidium spp, and in particular C. parvum, hasproved to be one of the commonest etiological agents of neonataldiarrhoea, which causes severe growth retardation and without suitabletreatment may be fatal to the animal.

Neonatal diarrhoea constitutes a constant threat for breeders ofbovines, ovines and caprins, and represents a considerable economic lossdue to the loss of income resulting from retardation of growth,mortality of neonates, intervention of veterinarians and the costs oftherapeutic treatment and rehydration. The etiology of neonataldiarrhoea is often multiple, several infectious agents in fact beingresponsible for these symptoms, in particular rotaviruses,coronaviruses, BVDV (Bovine Viral Diarrhoea Virus), Escherichia coliand, of course, C. parvum. The role of these various infectious agentsin neonatal diarrhoea of ruminants is difficult to estimate asdiagnostics is rarely carried out and there are regularly multipleinfections. However, C. parvum is currently considered to be the mainfactor in neonatal diarrhoea (de Graaf et al., 1999, Int. J. Parasitol.,29: 1269-1287). Thus, a study carried out on calves with neonataldiarrhoea demonstrated that 40% of them were infected with C. parvum andthree-quarters of them did not have any other infectious agent (datafrom “Veterinary and Agrochemical Research Centre” in Brussels).

In order to reduce the intensity and duration of the symptoms, numerousmolecules have been evaluated in various models, but none has givensatisfactory results. Thus, in domestic ruminants, only halofuginonelactate (HaloCur®) and paromycin have produced useful results, but donot allow complete control of the parasite (Chartier, 2002, Le Pointvétérinaire Pathologic ovine et caprine [The veterinary perspective.Pathologies of sheep and goats], 112-117). In humans, and especially fortreating AIDS patients, the usual treatment is based on the use ofparomomycin (Humatin) or nitazoxanide. The two molecules have similarefficacy but nitazoxanide is judged less toxic. Moreover, specificinhibitors of calcium-dependent protein kinases of T. gondii and of C.parvum have recently been developed (WO 2011/094628 A1). These are inparticular compounds belonging to the classes of the pyrazolopyrimidinesand the imidazo[1,5-α]pyrazines. These inhibitors affect the capacity ofthe parasites for invasion and proliferation, without disturbing thebiological activities of the host cell.

In conclusion, whether in humans or animals, at present there is nospecific treatment making it possible to effectively combatcryptosporidiosis, and only prevention, based on strict rules ofhygiene, allow a reduction in the ingestion of water or foodstuffscontaminated with C. parvum.

There is consequently a real need for agents for the prevention andtreatment of cryptosporidiosis in mammals, in particular mammals thathave an immature or deficient immune system. In fact, as infection mayoccur during the first few days, the neonates have an immune system thatis still developing, making a conventional vaccination scheme impossiblein young mammals. Thus, prophylactic trials conducted with killedparasites on neonate calves in a region where the disease is highlyendemic did not show any significant protection (Harp et al., 1996,Am.J.Vet.Res., 57: 1586-1588; Harp et al., 1998, J.Dairy.Sci. 81:289-294).

It has, however, been demonstrated that the immune response to C. parvumleads to the development of a protective response of type Th1 withconsiderable production of the cytokines IL-12 and IFN-γ. It has alsobeen demonstrated that these cytokines are able to lessen the impact ofinfection (Lacroix et al., 2001, Infect. Immun., 69: 1635-42).

One of the aims of the invention is to provide an immunostimulantcapable of inducing a non-specific immune response in the neonate, withthe production of IL-12 and IFNγ.

Another aim of the invention is to provide an immunostimulant capable oflimiting the effects of infection of mammals, human or animal, byCryptosporidium parvum.

Yet another aim of the invention is to provide a prophylactic treatment,and in particular a vaccine, against cryptosporidiosis.

The present invention relates to strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp, isolated from their natural environmentand having an immunostimulant effect, for the use thereof in theprevention or the treatment, in a mammal, of a disorder associated withan apicomplexan of the family Cryptosporidiidae.

The present invention relates to attenuated strains of Sarcocystidaeselected from attenuated Toxoplasma spp or attenuated Neospora spp,isolated from their natural environment and having an immunostimulanteffect, for the use thereof in the prevention or the treatment, in amammal, of a pathology associated with an apicomplexan of the familyCryptosporidiidae.

The present invention relates to strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, for the use thereof in theprevention or the treatment, in a mammal, of a pathology associated withan apicomplexan of the family Cryptosporidiidae, said strains havingattenuated virulence relative to the wild strains that induce apathology associated with Toxoplasma spp or Neospora spp.

By way of example, without limiting the scope of the present invention,such wild strains of apicomplexans may be illustrated by a virulentstrain of T. gondii of type RH for toxoplasmosis, or a virulent strainof N. caninum of type NC1 for neosporosis.

The present invention relates to strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, for the use thereof in theprevention or the treatment, in a mammal, of a pathology associated withan apicomplexan of the family Cryptosporidiidae, said strains havingattenuated virulence relative to a virulent strain, i.e. a strain havingvirulence substantially identical to the virulence of the strain fromwhich the strain with attenuated virulence was obtained.

By “immunostimulant” is meant the ability of a strain of Toxoplasma sppor Neospora spp to induce early activation of the immune system of thehost. This activation involves components of the immune system such asinterferons, cytokines, phagocytic cells, NK (Natural Killer) cells,dendritic cells and the complement system, which will act in anon-specific fashion on the targeted pathogen. This immunostimulation isnot based on the establishment of adaptive immunity.

By “prevention” is meant prophylaxis with the aim of preventing theappearance or spreading of a disease. It is in particular a question ofprotecting an individual with predisposition to contracting anddeveloping a disorder associated with an apicomplexan of the familyCryptosporidiidae. Such individuals are in particular neonates that havean immature immune system or individuals with an immune systemdysfunction. It is also a question of protecting a mammal exposed to arisk of contamination from its environment.

By “treatment” is meant not only inhibition of the progression of thepathology but also attenuation of the symptoms associated with thispathology. The treatment has the aim of reducing the extent of thesymptoms until they disappear completely, allowing the individual torecover a normal physiological state.

By “mammal” is meant human beings, certain commercial or farm animalsand certain pet animals.

By “attenuated strains of Toxoplasma spp or Neospora spp” is meantstrains of Toxoplasma spp or Neospora spp having an attenuatedvirulence, less than the virulence of the wild strains of T. gondii orof N. caninum capable of inducing a pathology, but which neverthelessconserve immunogenicity so as to be able to be used in the prevention orthe treatment of a disorder associated with an apicomplexan of thefamily Cryptosporidiidae. The attenuation of virulence may result eitherfrom a natural process of evolution of the species or may be induced inparticular by techniques of molecular biology familiar to a personskilled in the art. Whether it is of natural origin or the result ofhuman activity, the attenuation of virulence is due to the absence ofexpression of virulence factors or to the expression of one or morevirulence factors that are non-functional or have an altered function.The in vitro modification of the genetic heritage of Toxoplasma spp orNeospora spp confers a mutant character on the strain, relative to thewild-type strain from which it is derived. The wild strains of parasitesnot only have an immunogenic potential but are also virulent, i.e. theyare capable of inducing a pathology associated with Toxoplasma spp orNeospora spp (i.e. toxoplasmosis and neosporosis respectively), makingtheir use unsuitable in the context of the present invention. Thevirulence of the strains of Toxoplasma spp or of Neospora spp may inparticular be evaluated by in vitro cellular infectivity tests or byinfectivity tests in animals.

The cellular infectivity tests are carried out by depositing tachyzoiteson confluent cells, for example HFF (Human Foreskin Fibroblast) cells orVero cells, cell lines frequently used for the production of tachyzoitesof T. gondii or of N. caninum. The number of vacuoles formed and thenumber of parasites in each vacuole is determined by microscopicobservation. For the infectivity tests in animals, the various strainsare injected into the animals and the survival of these animals ismonitored over time (Cérède et al., 2005).

By “pathologies associated with an apicomplexan of the familyCryptosporidiidae” is meant the diseases resulting from an infection bya protozoon belonging to the phylum of the apicomplexans, and inparticular parasites belonging to the family Cryptosporidiidae, whichcomprises the genus Cryptosporidium. Several tens of species ofCryptosporidium are referenced (Fayer R, 2010, Exp. Parasitol., 124,90-7). These parasites are capable of invading the mucosal epithelia.

According to a particular embodiment, in the use according to thepresent invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said mammal is a neonate.

The present invention relates to strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp, isolated from their natural environmentand having an immunostimulant effect, for use in the prevention or thetreatment, in a neonate mammal, of a pathology associated with anapicomplexan of the family Cryptosporidiidae.

By “neonate” is meant a mammal from the time of its birth until it isweaned, i.e. until the moment when the mammal becomes capable of feedingitself and is no longer dependent on its mother's milk. The mainadvantage of using the mutant strain of Toxoplasma spp or Neospora sppfor the prevention and/or the treatment of pathologies associated withan apicomplexan of the family Cryptosporidiidae in the neonate isstimulation of its immature immune system in order to induce anon-specific immune response by synthesizing molecules that inhibit thegrowth of the Apicomplexa.

According to a particular embodiment, in the use according to thepresent invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said mammal is a human being or ananimal.

The animals to which the present invention relates are mainly commercialor farm animals, which are of interest to the agricultural and foodindustries, but also certain pet animals.

According to a more particular embodiment, in the use according to thepresent invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said animal belongs to the groupcomprising or constituted by of ovines, caprids, porcines, bovines,equines, camelids, canids or felids (Fayer, 2004, Vet. Parasitol., 126,37-59).

According to another embodiment, in the use according to the presentinvention of the strains of Sarcocystidae selected from Toxoplasma sppor Neospora spp isolated from their natural environment and having animmunostimulant effect, said strains of Toxoplasma spp or of Neosporaspp have at least an adhesin MIC1 and/or an adhesin MIC3 inactivated bya genetic modification relating to at least one of the genes mid and/ormic3.

By “an adhesin MIC1 and/or an adhesin MIC3” is meant the proteins of themicronemes, also called adhesins, MIC1 and/or MIC3 which play a role inthe mobility, the migration or the invasion of parasites of the phylumApicomplexa in its host. These proteins have linkage modules that allowthem to bind to the target cells of the host.

By “an inactivated adhesin” is meant an adhesin the function of whichcan no longer be ensured within the cell. An adhesin is inactivated whenit is not produced or when it is produced but does not have functionalactivity or has reduced functional activity. The inactivation alsorelates to an adhesin that can no longer bind to other proteins in orderto form a complex.

By “genetic modification” is meant any mutation made in the nucleic acidsequence of a gene leading to the absence of expression of the proteinencoded by that gene or leading to expression of a non-functional orless functional form of the protein encoded by that gene. This operationrequires human intervention when it is carried out in vitro. Thismutation may consist of the deletion of all or part of the gene, or ofits coding region, or of its promoter region, and of the insertion orthe substitution of nucleotides in the nucleotide sequence of the gene.

By “mic1 gene” is meant the gene coding for the protein of themicronemes MIC1, also called adhesin MIC1. This protein contains severalmodules, including binding domains that bind lactose specifically. Theprotein MIC1 is also capable of binding to the surface of the hostcells.

The detailed construction of the Toxo mid KO strain is described indocuments U.S. Pat. No. 7,946,185 B2 and EP 1 703 914 B1. The detailedconstruction of the Neo ncmic1 KO strain is described in the presentapplication.

By “mic3 gene” is meant the gene coding for the protein of themicronemes MIC-3, also called adhesin MIC3. This protein homodimerizesin order to form a complex of 90 kDa. MIC3 comprises domains of the EGFtype and a domain of the lectin type. The protein MIC3 is also capableof binding to the surface of the host cells.

The detailed construction of the Toxo mic3 KO strain is described indocuments U.S. Pat. No. 7,946,185 B2 and EP 1 703 914 B1. The detailedconstruction of the Neo ncmic3 KO strain is described in the presentapplication.

According to another particular embodiment, in the use according to thepresent invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said strains of Toxoplasma spp orof Neospora spp have the two adhesins MIC1 and MIC3 inactivated by agenetic modification relating to the two genes mid and mic3.

By “genetic modification relating to the two genes mid and mic3” ismeant the mutation made in the nucleic acid sequence of the mid gene andin that of the mic3 gene. This double mutation leads to the absence ofexpression of the proteins MIC1 and MIC3 or leads to the expression of anon-functional or less functional form of the proteins MIC1 and MIC3.These mutant strains of Toxoplasma spp or of Neospora spp are calledToxo mic1-3 KO or Neo ncmic1-3 KO respectively and have a veryattenuated virulence in comparison with the wild strains of T. gondii oftype RH or of N. caninum of type NC1 from which they are derived.However, the Toxo mic1-3 KO or Neo ncmic1-3 KO strains retain a strongimmunogenicity. The detailed construction of the Toxo mic1-3 KO strainis described in documents U.S. Pat. No. 7,946,185 B2 and EP 1 703 914B1. The detailed construction of the Neo ncmic1-3 KO strain is describedin the present application.

The Toxo mic1-3 KO and Neo ncmic1-3 KO strains retained their capacityfor colonizing the target tissues without the development of apathogenic effect following administration of said strains to a mammal.The knock out of the genes mid and mic3 has little or no effect on theimmunogenic potential of these strains, but reduces their virulenceconsiderably relative to a virulent strain, i.e. a strain having avirulence substantially identical to the virulence of the strain fromwhich the strain with attenuated virulence was obtained.

The inoculation of the Toxo mic1-3 KO strain in neonate mice leads tothe production of cytokines IL-12 and IFN-γ and effectively protects themouse pups from subsequent infection with Cryptosporidium parvum.

The mutant Toxo mic1-3 KO strain effectively stimulates the productionof IL-12 and IFN-γ from cells of the mesenteric lymph nodes andsplenocytes of lambs, confirming the possibility of using this mutantstrain as an immunostimulant in this target animal species.

According to a particular embodiment, in the use according to thepresent invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said strains of Toxoplasma spp orof Neospora spp are respectively Toxoplasma gondii or Neospora caninum.

According to another embodiment, in the use according to the presentinvention of the strains of Sarcocystidae selected from Toxoplasma sppor Neospora spp isolated from their natural environment and having animmunostimulant effect, said immunostimulant effect of said strainsleads to the secretion of interleukin-12 (IL-12) and then ofinterferon-γ (IFN-γ).

By “interleukin-12 (IL-12)” is meant the cytokine synthesized by immunesystem cells such as the monocytes, the dendritic cells and themacrophages. The cytokine is secreted early and will thus activate thetarget cells (T cells and Natural Killer cells) so that the lattersecrete IFN-γ in their turn.

By “interferon-γ (IFN-γ)” is meant the cytokine synthesized by the Tlymphocytes CD4+, CD8+ and the Natural Killer (NK) cells activated byIL-12. The secretion of IFN-γ by the cells of the organism will allowproduction of an innate response protective against C. parvum. Thiscytokine will have a pleiotropic activity owing to the diversity of thetargeted cell lines.

By “cytokines” is meant all the molecules involved in the developmentand the regulation of the immune system. The cytokines are glycosylatedor non-glycosylated proteins, which may be classified according to theirbiological activity:

-   -   pro-inflammatory: this includes the interleukins (IL) and the        tumour necrosis factors (TNF),    -   immunoregulatory: this includes the interleukins (IL),    -   effector: this includes the interferons (IFN), the tumour        necrosis factors (TNF) and the chemokines.

According to another more particular embodiment, in the use according tothe present invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said secretion of interleukin-12(IL-12) and of interferon-γ (IFN-γ) begins between 3 and 9 days afterusing said strains of Toxoplasma spp or of Neospora spp asimmunostimulant.

According to a more particular embodiment, in the use according to thepresent invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said pathology associated with anapicomplexan of the family Cryptosporidiidae is cryptosporidiosis.

By “cryptosporidiosis” is meant the pathology characterized by symptomssuch as cramps, fever, fatigue, nausea and especially diarrhoea, whichmay lead to the dehydration of the mammal infected by Cryptosporidiumparvum or by another Cryptosporidium species. Cryptosporidiosis mainlyaffects the intestines of mammals but may also infect the biliary andpancreatic tracts and the respiratory tract in an immunodepressedindividual. The infestation occurs by ingestion of oocysts present inthe excrement of parasitized animals with which the environment iscontaminated (water, earth or raw food). The symptoms appear two to tendays after the infection occurred and persist for more than two weeks.The severity of this pathology varies depending on the age of theinfected host but more particularly on the state of its immune system.In a host having a mature and functional immune system,cryptosporidiosis has no effect on the health of the host. Conversely, ahost that is very young, very old or has an immature or deficient immunesystem may develop very severe forms of cryptosporidiosis.

By “immature or deficient immune system” is meant the immune system forwhich one or more cell lines are either absent, or deficient. Theimmaturity of the immune system is a common trait in neonate mammals,which makes them particularly vulnerable to parasitic infections. Theabsorption of antibodies from the mother via the colostrum endows theneonate with a certain degree of protection while its immune system isbeing established. The immaturity or the deficiency of the immune systemmay also result from pathologies of a genetic origin, in particular inhumans, such as Wiskott-Aldrich syndrome, an X-linkedlymphoproliferative syndrome. This deficiency may also arise after aninfection by the human immunodeficiency virus (HIV) or following atherapeutic treatment associated with an organ graft or the medicalmanagement of certain cancers by chemo- or radiotherapy. The mammalsthat have an immature or deficient immune system are particularlyvulnerable to infections with apicomplexans.

According to an even more particular embodiment, in the use according tothe present invention of the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, said apicomplexan of the familyCryptosporidiidae responsible for cryptosporidiosis is at least oneapicomplexan selected from the group constituted by Cryptosporidiumparvum, Cryptosporidium bovis, Cryptosporidium andersoni,Cryptosporidium ryanae, Cryptosporidium muris, Cryptosporidiumubiquitum, Cryptosporidium hominis, Cryptosporidium canis,Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidiummeleagridis or Cryptosporidium xiaoi.

By “Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidiumandersoni, Cryptosporidium ryanae, Cryptosporidium muris,Cryptosporidium ubiquitum, Cryptosporidium hominis, Cryptosporidiumcanis, Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidiummeleagridis or Cryptosporidium xiaoi” is meant the protozoa of thephylum Apicomplexa capable of causing intestinal pathologies of a hydricnature in the majority of mammals. These parasites are in particularcapable of causing cryptosporidiosis in ovines, caprins, porcines,bovines, equines, camels, camelids, canids, felids or humans (Payer,2004, Vet. Parasitol., 126, 37-59) that have an immature or deficientimmune system.

The present invention also relates to the strains of Toxoplasma gondiior of Neospora caninum that have the two adhesins MIC1 and MIC3inactivated by a genetic modification relating to the two mid and micagenes for the use thereof in the prevention or the treatment, in amammal, of a pathology associated with an apicomplexan of the familyCryptosporidiidae, said pathology associated with an apicomplexan of thefamily Cryptosporidiidae being cryptosporidiosis.

The present invention also relates to the strains of Toxoplasma gondiior of Neospora caninum isolated from their natural environment andhaving an immunostimulant effect, for use in the prevention or thetreatment, in a mammal, of a pathology associated with an apicomplexanof the family Cryptosporidiidae, said strains of Toxoplasma gondii or ofNeospora caninum having the two adhesins MIC-1 and MIC-3 inactivated bya genetic modification relating to the two mic-1 and mic-3 genes, andsaid pathology associated with an apicomplexan of the familyCryptosporidiidae is cryptosporidiosis.

The invention also relates to the strains of Toxoplasma gondii or ofNeospora caninum isolated from their natural environment and having animmunostimulant effect, said strains of Toxoplasma gondii or of Neosporacaninum having the two adhesins MIC1 and MIC3 inactivated by a geneticmodification relating to the two mic1 and mic3 genes for the use thereofin the prevention or the treatment, in a mammal, of a pathologyassociated with an apicomplexan of the family Cryptosporidiidae, saidstrains of Toxoplasma gondii or of Neospora caninum being administeredto said mammal at a rate from 20 to 10⁹ tachyzoites.

By “tachyzoite” is meant the rapidly multiplying form of Toxoplasmagondii or of Neospora caninum. The tachyzoite has a crescent shape and avariable size of 5-8×2-3 μm. The apical part of the parasite comprisesconoids which participate in the penetration of the parasite into thehost cell. The micronemes, the rhoptries and the dense granulesconstitute the three major organelles of the tachyzoite, which alsocomprises a nucleus, an apicoplast, a Golgi apparatus, an endoplasmicreticulum and an organite similar to the mitochondrion.

The determination of an effective dose of tachyzoites for theprophylactic treatment of mammals makes it possible to limit theinfection or the transmission of the pathogenic agent responsible forcryptosporidiosis. Such a treatment may be adapted and/or repeated asmany times as necessary by a person skilled in the art, depending on theage and immunological status of the mammal.

The tachyzoites may be brought into contact with the mammal not only ona mammal presenting the symptoms characteristic of cryptosporidiosis butalso on a mammal without any of the symptoms of cryptosporidiosis butwhich is in contact with other mammals infected by Cryptosporidiumparvum or by another species of Cryptosporidium able to inducecryptosporidiosis.

According to a particular embodiment, the strains of Sarcocystidaeselected from Toxoplasma spp or Neospora spp isolated from their naturalenvironment and having an immunostimulant effect for the use thereofaccording to the present invention are in a galenic form selected fromthe group comprising or constituted by liquid suspensions, solid orliquid dispersions, powders, pastes or lyophilizates.

The galenic form is adapted by a person skilled in the art depending onthe method of administration selected. All the conventional methods ofadministration may be envisaged: by parenteral route (intravenous,subcutaneous, intradermal, intramuscular, intraperitoneal, andintranasal) or by enteral route.

According to a more particular embodiment, the strains of Sarcocystidaeselected from Toxoplasma spp or Neospora spp isolated from their naturalenvironment and having an immunostimulant effect for the use thereofaccording to the present invention are associated with at least oneother antigen, or at least one adjuvant, or at least one stabilizer, orat least one preservative or a mixture of at least two of said productsfor increasing the immune response of said mammal.

By “antigen” is meant any natural or recombinant protein, in its nativeor mutated form, originating from a parasite or from a pathogen otherthan Toxoplasma spp or Neospora spp capable of inducing a cellular orhumoral immune response in a mammal. The aim of combining the mutantstrain of Toxoplasma spp or Neospora spp with such an antigen is toamplify the mammal's immune response and thus endow it with betterprotection against an apicomplexan infection.

By “adjuvant” is meant any substance capable of reinforcing andprolonging the immune response directed against the targeted antigen.The mechanism involved in order to make the immune response moreeffective is dependent on the adjuvant used. The adjuvants aresubstances which are well known to a person skilled in the art and inparticular include aluminium salts, squalene, saponins, the bacterialconstituents or toxins, or also certain proteins (peptone, albumin,casein).

By “stabilizers or preservatives” is meant the compounds allowingperfect preservation of the strains of Toxoplasma spp or Neospora spp intheir packaging.

Stabilizers or preservatives are substances which are well known to aperson skilled in the art and in particular include the carbohydrates(sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), thepolar organic solvents, such as DMSO (dimethylsulphoxide), and thepolysorbates.

By “to increase the immune response” is meant the activation of variouspathways of the immune system. In a mammal that has an immature ordeficient immune system, it is a question of activating the non-specificresponse by increasing the synthesis of different categories ofcytokines, such as the interferons or the interleukins. Among thesecytokines interleukin-12 (IL-12) may be mentioned, which will activatethe CD4+ or CD8+LT cells as well as the NK cells. These activated cellswill in their turn secrete interferon γ (IFN-γ), which plays a role inthe immunological mechanisms that control the multiplication of manyintracellular parasites, thus preventing their propagation in theorganism. In an adult mammal having a mature immune system, the increasein the immune response passes via an adaptive immunity (specificproliferation in response to the foreign antigens) in addition to thenon-specific response.

The present invention also relates to a method of inducing an immuneresponse in a mammal comprising a step of administration, to saidmammal, of tachyzoites of strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp, isolated from their natural environmentand having an immunostimulant effect, allowing an immune response to beinduced.

The present invention also relates to a method of inducing an immuneresponse in a neonate mammal comprising a step of administration, tosaid neonate mammal, of tachyzoites of strains of Sarcocystidae selectedfrom Toxoplasma spp or Neospora spp, isolated from their naturalenvironment and having an immunostimulant effect, allowing an immuneresponse to be induced.

By “immune response” is meant the two essential phases constituted bythe recognition of the antigen and the reaction intended to eliminatesaid antigen. When an antigen enters the body for the first time, it isthe non-specific immune response that is activated. It uses non-clonalmechanisms, since it does not require specific cellular clones. Theinflammatory reaction created by this non-specific response will lead toadaptive immunity. This response involves cells specifically recognizingthe antigen and will then lead to clonal expansion. This clonalexpansion will result in a very effective immune response and a memoryresponse (specific immune response following the second entry of theantigen into the organism).

According to a particular embodiment, in the method according to thepresent invention, said mammal is a neonate.

According to a more particular embodiment, in the method according tothe present invention, said mammal is a human being or an animal.

According to an even more particular embodiment, in the method accordingto the present invention, the animal belongs to the group comprising orconstituted by ovines, caprins, porcines, bovines, equines, camelids,canids or felids.

According to another embodiment, in the method according to the presentinvention, said strains of Sarcocystidae selected from Toxoplasma spp orNeospora spp have at least one adhesin MIC1 and/or one adhesin MIC3inactivated by a genetic modification which relates to at least one ofthe mic1 and/or mic3 genes.

According to another particular embodiment, in the method according tothe present invention, said strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp have the two adhesins MIC1 and MIC3inactivated by a genetic modification which relates to the two mid andmic3 genes.

According to another more particular embodiment, in the method accordingto the present invention, said strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp are respectively Toxoplasma gondii orNeospora caninum.

According to yet another particular embodiment, in the method accordingto the present invention, the strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp induce a non-specific immune response inparticular characterized by the secretion of IL-12 and/or IFN-γ.

The present invention also relates to a method for protecting a mammalagainst a parasitosis associated with an apicomplexan of the familyCryptosporidiidae comprising a step of administration, to said mammal,tachyzoites of strains of Sarcocystidae selected from Toxoplasma spp orNeospora spp, isolated from their natural environment and having animmunostimulant effect, for inducing protection against saidparasitosis.

According to a more particular embodiment, in the method according tothe present invention, the mammal is a neonate.

According to an even more particular embodiment, in the method accordingto the present invention, the mammal is a human being or an animal.

According to a particular embodiment, in the method according to thepresent invention, the animal belongs to the group comprising orconstituted by ovines, caprins, porcines, bovines, equines, camelids,canids or felids.

According to a particular embodiment, in the method according to thepresent invention, said strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp have at least one adhesin MIC1 and/or oneadhesin MIC3 inactivated by a genetic modification which relates to atleast one of the mic1 and/or mic3 genes.

According to a more particular embodiment, in the method according tothe present invention, said strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp have the two adhesins MIC1 and MIC3inactivated by a genetic modification which relates to the two mid andmic3 genes.

According to a more particular embodiment, in the method according tothe present invention, said parasitosis associated with an apicomplexanof the family Cryptosporidiidae is cryptosporidiosis.

According to another more particular embodiment, in the method accordingto the present invention, the apicomplexan of the familyCryptosporidiidae responsible for the cryptosporidiosis is at least oneapicomplexan selected from the group constituted by Cryptosporidiumparvum, Cryptosporidium bovis, Cryptosporidium andersoni,Cryptosporidium ryanae, Cryptosporidium muris, Cryptosporidiumubiquitum, Cryptosporidium hominis, Cryptosporidium canis,Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidiummeleagridis or Cryptosporidium xiaoi.

According to an even more particular embodiment, in the method accordingto the present invention, the administration, to said mammal, of saidtachyzoites of said strains of Sarcocystidae selected from Toxoplasmaspp or Neospora spp is carried out by enteral route or by parenteralroute.

According to yet another embodiment, in the method according to thepresent invention, the administration, to said mammal, of saidtachyzoites of said strains of Sarcocystidae selected from Toxoplasmaspp or Neospora spp is carried out before exposure of said mammal to theapicomplexan of the family Cryptosporidiidae responsible forcryptosporidiosis.

According to yet another embodiment, in the method according to thepresent invention, the administration, to said mammal, of saidtachyzoites of said strains of Sarcocystidae selected from Toxoplasmaspp or Neospora spp is carried out during exposure of said mammal to theapicomplexan of the family Cryptosporidiidae responsible forcryptosporidiosis.

According to yet another embodiment, in the method according to thepresent invention, the administration, to said mammal, of saidtachyzoites of said strains of Sarcocystidae selected from Toxoplasmaspp or Neospora spp is carried out after exposure of said mammal to theapicomplexan of the family Cryptosporidiidae responsible forcryptosporidiosis.

In the aforementioned three embodiments, said mammal may be a neonate.

The following figures and examples are given purely by way ofillustration of the subject of the present invention and do not in anyway constitute a limitation thereof.

DESCRIPTION OF THE FIGURES

FIG. 1: this figure illustrates the 2 steps of homologous recombinationin order to obtain the strain Neo ncmic1-3 KO. The first step ofhomologous recombination allows integration of the gene coding for theenzyme dihydrofolate reductase (DHFR) at the locus of the ncmic3 gene.Selection with pyrimethamine makes it possible to amplify the mutantsingle strain Neo ncmic3 KO. The Neo ncmic3 KO strain thus obtainedserves for the second step of homologous recombination, which allows theintegration of the gene coding for the chimeric proteinchloramphenicol-acetyl-transferase/green fluorescent protein (CAT-GFP)at the locus of the ncmic1 gene. Selection with chloramphenicol thenmakes it possible to amplify the double mutant strain Neo ncmic1-3 KO.

FIG. 2-A: this figure is a schematic representation of thepNcMic3KO-DHFR plasmid. This plasmid with 11,312 base pairs comprisesthe DHFR selection gene flanked by the homologous regions (5HR-NcMic3and 3HR-NcMic3) of the sequences flanking the ncmic3 gene, theampicillin resistance gene (Amp) as well as the Not I restriction sitewhich permits its linearization.

FIG. 2-B: this figure is a schematic representation of thepNcMic1KO-CAT-GFP plasmid. This plasmid with 10,069 base pairs comprisesthe CAT-GFP selection gene flanked by the homologous regions (3HR-NcMic1and 5HR-NcMic1) of the sequences flanking the ncmic1 gene, theampicillin resistance gene (Amp) as well as the Kpn I restriction sitewhich permits its linearization.

FIG. 3-A: this figure shows the electrophoretic profiles of the PCRproducts obtained respectively in the wild-type strain NC1 of N. caninumand in the mutant strain Neo ncmic3 KO, using the sets of PCR primersNo. 1, No. 2 or No. 3 in Table II which correspond to SEQ ID NO: 5 toSEQ ID NO: 10.

FIG. 3-B: this figure shows the electrophoretic profiles of the PCRproducts obtained respectively in the wild-type strain NC1 of N. caninumand in the mutant strain Neo ncmic3 KO, using the sets of PCR primersNo. 4, No. 5, No. 6 or No. 7 in Table II which correspond to SEQ ID NO:11 to SEQ ID NO: 16.

FIG. 4-A: this figure illustrates the analysis for detecting the NcMIC3protein in the wild-type strain NC1 of N. caninum by immunofluorescence,using an antibody specifically recognizing the NcMIC3 protein. One andthe same microscopic field is visualized in direct light (image A) or influorescence (image B).

FIG. 4-B: this figure illustrates the analysis for detecting the NcMIC3protein in the mutant strain of N. caninum Neo ncmic3 KO byimmunofluorescence, using an antibody specifically directed against theNcMIC3 protein. One and the same microscopic field is visualized indirect light (image A) or in fluorescence (image B).

FIG. 5: this figure shows the electrophoretic profiles of the PCRproducts obtained respectively in the wild-type strain NC1 of N.caninum, in the mutant strain Neo ncmic3 KO and in the mutant strain Neoncmic1-3 KO using the sets of PCR primers No. 1 to No. 12 in Table VIIwhich correspond to SEQ ID NO: 7 to 16 and to SEQ ID NO: 21 to 30.

FIG. 6: this figure illustrates the analysis for detecting the GFPprotein in the mutant strains Neo ncmic3 KO (images A and B) and Neoncmic1-3 KO (image C and D) by immunofluorescence, using the fluorescentproperties of the CAT-GFP protein. One and the same microscopic field isvisualized in direct light (top images A and C) or in fluorescence(bottom images B and D).

FIG. 7: FIG. 7 illustrates the assay of antibodies of type IgManti-Toxoplasma gondii in mouse pup serum. The assay of antibodies oftype IgM anti-Toxoplasma gondii in the serum was carried out 3 days(empty grey squares) or 9 days (filled black squares) after the mousepups had received 20 tachyzoites of the strain Toxo mic1-3 KO byintraperitoneal route. The mouse pups that had not received anytachyzoite of the strain Toxo mic1-3 KO served as controls.

-   -   on the x-axis: batches of C57BL/6 mouse pups controls (A) and        inoculated with the strain Toxo mic1-3 KO (B)    -   on the y-axis: absorbance values measured at 405 nm.        The three mouse pups that received 20 tachyzoites of the strain        Toxo mic1-3 KO and were sacrificed at 9 days are shown by their        number (4, 5, 6) in FIG. 7.

FIG. 8-A: FIG. 8-A illustrates the measurement of the expression ofinterferon-gamma (IFN-γ) in the ileum of the mouse pup. The expressionlevel of the gene coding for IFN-γ was measured by quantitative PCR onthe total RNAs extracted from a fragment of ileum using specific primers(SEQ ID NO: 33 to 34), 9 days after the mouse pups had received 20tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route(filled black squares). The mouse pups that had not received anytachyzoites of the strain Toxo mic1-3 KO served as controls (empty greysquares).

-   -   on the x-axis: batches of C57BL/6 mouse pups controls (A) and        inoculated with the strain Toxo mic1-3 KO (B)    -   on the y-axis: IFN-γ/HPRT ratio.        The three mouse pups that received 20 tachyzoites of the Toxo        mic1-3 KO strain and were sacrificed at 9 days are shown by        their number (4, 5, 6) in FIG. 8-A.

FIG. 8-B: FIG. 8-B illustrates the measurement of the expression ofinterleukin-12 (p40 subunit) (IL-12p40) in the mouse pup ileum. Theexpression level of the gene coding for IL-12p40 was measured byquantitative PCR on the total RNAs extracted from a fragment of ileumusing specific primers (SEQ ID NO: 31 to 32), 9 days after the mousepups received 20 tachyzoites of the strain Toxo mic1-3 KO byintraperitoneal route (filled black squares). The mouse pups that hadnot received any tachyzoites of the strain Toxo mic1-3 KO served ascontrols (empty grey squares):

-   -   on the x-axis: batches of C57BL/6 mouse pups controls (A) and        immunostimulated by the strain Toxo mic1-3 KO (B)    -   on the y-axis: IL-12p40/HPRT ratio.        The three mouse pups that received 20 tachyzoites of the strain        Toxo mic1-3 KO and were sacrificed at 9 days are shown by their        number (4, 5, 6) in FIG. 8-B.

FIG. 9: FIG. 9 illustrates the detection of the presence of tachyzoitesof Toxo mic1-3 KO in the intestine of the mouse pup. The detection ofthe presence of tachyzoites of Toxo mic1-3 KO was carried out byimmunohistology on sections of the intestine 9 days after the mouse pupsreceived 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitonealroute Immunolabelling is carried out using a rabbit anti-SAG-1polyclonal antibody and an anti-rabbit secondary antibody coupled tofluorescein isothiocyanate.

The detection of tachyzoites of Toxoplasma gondii mic1-3 KO is shown forthe intestines of the mouse pups No. 4 (B) and No. 6 (D). Thetachyzoites appear in the form of white dots in the intestinal musclesof the mouse pup No. 4 (B).

The nuclei of the cells of the intestinal villi and intestinal musclesof the mouse pups No. 4 (C) and No. 6 (E) are labelled with Hoechst andappear in the form of white dots.

FIG. 10: FIG. 10 illustrates the presence of Toxoplasma gondii via theexpression of the surface antigen SAG-1 in the ileum of the mouse pup.

The cDNAs obtained from the total RNAs extracted from a fragment ofileum served as a matrix for the amplification of SAG-1 by PCR usingspecific primers (SEQ ID NO: 37 to 38). The mouse pups were sacrificedthree days (T1) or 9 days (T2) after receiving 20 tachyzoites of thestrain Toxo mic1-3 KO by intraperitoneal route (V). The mouse pups thathad not received any tachyzoite of the strain Toxo mic1-3 KO served ascontrols (NV). The PCR products were deposited on agarose gel.

SAG-1 was also amplified from the genomic DNA of the wild-type strain ofToxoplasma gondii (RH) and from the genomic DNA of the mutated strain ofToxo mic1-3 KO (KO). The amplification products serve as controls of thesize of the amplification product of SAG-1.

The mouse pups are shown by their number (1, 2, 3, 4, 5, 6) in FIG. 10.

FIG. 11: FIG. 11 illustrates the count of the oocysts of Cryptosporidiumparvum in the intestine of the mouse pup. The number of oocysts ofCryptosporidium parvum was counted in a Thoma cell from an extract ofground material of the intestine placed in a sugar solution. Theintestines originate from:

-   -   the mouse pups challenged with 500 000 parasites of        Cryptosporidium parvum, which had received 20 tachyzoites of the        strain Toxo mic1-3 KO by intraperitoneal route 3 days prior the        challenge with Cryptosporidium parvum (filled black squares), or    -   the mouse pups challenged with 500 000 parasites of        Cryptosporidium parvum, which had not received any tachyzoite of        the strain Toxo mic1-3 KO (empty grey squares).

The mouse pups are sacrificed 6 days after the infection byCryptosporidium parvum.

-   -   on the x-axis: the batches of C57BL/6 mouse pups infected by        Cryptosporidium parvum alone (C.p) or by Toxo mic1-3 KO and then        Cryptosporidium parvum (T.g+C.p)    -   on the y-axis: the total number of oocysts of Cryptosporidium        parvum in the intestine.

FIG. 12: FIG. 12 illustrates the detection of the presence of oocysts ofCryptosporidium parvum in the intestine of the mouse pup. The number ofoocysts of Cryptosporidium parvum was counted in a Thoma cell from anextract of ground material of the intestine placed in a sugar solution.The intestines originate from:

-   -   the mouse pups challenged with 1 000 000 parasites of        Cryptosporidium parvum, which had received 20 000 tachyzoites of        the strain Toxo mic1-3 KO by oral route 3 days prior the        challenge with Cryptosporidium parvum (filled black squares), or    -   the mouse pups challenged with 1 000 000 parasites of        Cryptosporidium parvum, which had not received any tachyzoite of        the strain Toxo mic1-3 KO (empty grey squares).

The mouse pups are sacrificed 7 days after the infection byCryptosporidium parvum.

-   -   on the x-axis: the batches of C57BL/6 mouse pups infected by        Cryptosporidium parvum alone (C.p) or by Toxo mic1-3 KO and then        Cryptosporidium parvum (T.g+C.p)    -   on the y-axis: the total number of oocysts of Cryptosporidium        parvum in the intestine.

FIG. 13-A: FIG. 13-A illustrates the assay of interleukin-12 (IL-12) inthe mononuclear cells from the spleen of lambs and of adult sheep.Samples of mononuclear cells from the spleen of lambs (grey triangles)and of adult sheep (black squares) were infected in vitro by threedifferent strains of Toxoplasma gondii: type I wild-type strain (RH),type I mutant strain Toxo mic1-3 KO (KO) and type II wild-type strain(Pru). Mononuclear cells from the spleen of lambs and of adult sheep notinfected in vitro serve as controls (M).

-   -   on the x-axis: the “RH”: type I wild-type strain of Toxoplasma        gondii, “KO”: strain Toxo mic 1-3 KO, “Pru” type II wild-type        strain, “M”: spleen cells cultured in vitro without stimulant        (negative control)    -   on the y-axis: the concentration of IL-12 (IU/ml).

FIG. 13-B: FIG. 13-B illustrates the assay of interleukin-12 (IL-12) inthe mononuclear cells originating from the mesenteric lymph nodes oflambs and of adult sheep. Samples of mononuclear cells originating fromthe mesenteric lymph nodes of lambs (grey triangles) and of adult sheep(black squares) were infected in vitro by three different strains ofToxoplasma gondii: type I wild-type strain (RH), type I mutant strainToxo mic1-3 KO (KO) and type II wild-type strain (Pru). Mononuclearcells from the mesenteric lymph nodes of lambs and of adult sheep notinfected in vitro serve as controls (M).

-   -   on the x-axis: the “RH”: type I wild-type strain of Toxoplasma        gondii, “KO”: strain Toxo mic 1-3 KO, “Pru” type II wild-type        strain, “M”: spleen cells cultured in vitro without stimulant        (negative control)    -   on the y-axis: the concentration of IL-12 (IU/ml).

FIG. 13-C: FIG. 13-C illustrates the assay of interferon gamma (IFNγ) inthe mononuclear cells from the spleen of lambs and of adult sheep.Samples of mononuclear cells from the spleen of lambs (grey triangles)and of adult sheep (black squares) were infected in vitro by threedifferent strains of Toxoplasma gondii: type I wild-type strain (RH),type I mutant strain Toxo mic1-3 KO (KO) and type II wild-type strain(Pru). Mononuclear cells from the spleen of lambs and of adult sheep notinfected in vitro serve as controls (M).

-   -   on the x-axis: the “RH”: type I wild-type strain of Toxoplasma        gondii, “KO”: strain Toxo mic 1-3 KO, “Pru” type II wild-type        strain, “M”: spleen cells cultured in vitro without stimulant        (negative control)    -   on the y-axis: the concentration of IFNγ (ng/ml).

FIG. 14: FIG. 14 illustrates the change in body weight of the lambsimmunostimulated with 10⁶ tachyzoites Toxo mic1-3 KO, one day aftertheir birth (batch A-black square) in comparison with control lambs notimmunostimulated (batch B-grey circle).

-   -   on the x-axis: the time elapsed after stimulation of the lambs        by Toxo mic1-3 KO (in days)    -   on the y-axis: the body weight of the lambs (in kilograms).

FIG. 15: FIG. 15 illustrates the production of IFN-γ after restimulationwith the total extract of the strain Toxo mic1-3 KO of mononuclear cellsof the spleen of lambs immunostimulated with 10⁶ tachyzoites Toxo mic1-3KO, one day after their birth (lambs No. 1416-1418-1421 and 1424) or ofcontrol lambs (lambs 1428 and 1423), sacrificed 15 days after the immunostimulation,

-   -   on the x-axis: the “medium”: spleen cells cultured in vitro        without stimulant (control−), “Mic1-3 KO ET: spleen cells        restimulated with the total parasite extract of the strain Toxo        mic1-3 KO and “ConA”: spleen cells stimulated by concanavalin A        (control+)]    -   on the y-axis: the concentration of IFN-γ (in ng/ml).

FIG. 16: FIG. 16 illustrates the production of IFN-γ produced from theex vivo culture of cells of subiliac and popliteal lymph nodes of lambsimmunostimulated with 10⁶ tachyzoites Toxo mic1-3 KO, one day aftertheir birth (lambs No. 1416-1418-1421 and 1424) or of control lambs(lambs 1428 and 1423), sacrificed 15 days after the immunostimulation.

-   -   on the x-axis: the cells of popliteal lymph nodes (A) and cells        of subiliac lymph nodes (B)    -   on the y-axis: the concentration of IFN-γ (in pg/ml).

FIG. 17: FIG. 17 illustrates the survival of lambs immunostimulated with10⁶ tachyzoites Toxo mic1-3 KO, one day after their birth and challengedwith 5.10⁶ oocysts of C. parvum (black square) and the control lambsonly challenged (grey circle).

The survival curves are of the Kaplan-Meier type.

-   -   on the x-axis: the time elapsed after infection of the lambs        by C. parvum (in days)    -   on the y-axis: the survival of the lambs (in %).

FIG. 18: FIG. 18 illustrates the daily weight gain of lambsimmunostimulated with 10⁶ tachyzoites Toxo mic1-3 KO, one day aftertheir birth and challenged with 5.10⁶ oocysts of C. parvum (blacksquare) and the control lambs only challenged (grey diamond).

-   -   on the x-axis: the time elapsed after stimulation of the lambs        by Toxo mic1-3 KO (in days)    -   on the y-axis: the weight change of the lambs (in kg).

The average weight gain (DAG, daily average gain) reflects the rate ofincrease as a function of time according to the formula: DAG weight=dWeight/d′ Age.

FIG. 19: FIG. 19 illustrates the mean excretion of oocysts C. parvum pergram of excrement of lambs immuno stimulated with 10⁶ tachyzoites Toxomic1-3 KO, one day after their birth and challenged with 5.10⁶ oocystsof C. parvum (black square) and the control lambs only challenged (greydiamond).

-   -   on the x-axis: the time elapsed after infection of the lambs        by C. parvum (in days)    -   on the y-axis: number of oocysts of C. parvum per gram of        excrement.

EXPERIMENTAL SECTION

In order to prepare the strain of N. caninum with the ncmic1 and ncmic3genes knocked out, two steps of homologous recombination were carriedout. The first step of homologous recombination makes it possible toobtain a simple mutant KO (strain Neo ncmic3 KO). The second step ofhomologous recombination is carried out in the strain Neo ncmic3 KO inorder to obtain a doubly deleted strain (Neo ncmic1-3 KO) (FIG. 1).

Example 1 Construction of the Mutant Strain Neo Ncmic3 KO

The haploidy of the genome of Neospora caninum during the proliferativephase allows inactivation of a gene in a single homologousrecombination.

All the tachyzoites of the strain NC1 of Neospora caninum used wereproduced in human fibroblasts (HFF) cultured in Dulbecco's minimummedium (DMEM) supplemented with 10% of foetal calf serum (FCS), 2 mM ofglutamine, 50 U/mL of penicillin and 50 μg/mL of streptomycin. They wereharvested after mechanical lysis of the host cells and 3 passes througha 25 G syringe.

a) Construction of the Plasmid pNcMic3KO-DHFR

The plasmid pNcMic3KO-DHFR (FIG. 2-A) contains the DHFR (dihydrofolatereductase) selection gene, which confers resistance to pyrimethamine.The DHFR selection gene is placed under the control of the α-tubulinpromoter of Toxoplasma gondii (aTUB5 promoter) to allow expression ofthe gene in the parasite. The efficacy of this heterologous promoter hadbeen demonstrated beforehand in N. caninum. This cassette is framed bythe homologous regions (5HR-NcMic3 and 3HR-NcMic3) of the sequencesflanking the ncmic3 gene. The DHFR selection cassette makes it possibleto carry out selection for pyrimethamine.

The 5′UTR region of the ncmic3 gene was amplified by PCR from thegenomic DNA of the strain NC1 of Neospora caninum. For theamplification, the primers 5 HR NCmic3 F KpnI and 5 HR NCmic3 R ClaI(SEQ ID NO: 1 and SEQ ID NO: 2) allow amplification of the 5′UTR regionof the ncmic3 gene and creation of two restriction sites, which wereused for cloning the 5HR fragment upstream of the DHFR selectioncassette into the plasmid pT230 DHFR (KpnI at 5′ and ClaI at 3′ of thePCR fragment).

The 3′UTR region of the ncmic3 gene was amplified by PCR from thegenomic DNA of the strain NC1 of Neospora caninum. For theamplification, the primers 3 HR NCmic3 F XbaI and 3 HR NCmic3 R NotI(SEQ ID NO: 3 and SEQ ID NO: 4) allow amplification of the 3′UTR regionof the ncmic3 gene and creation of two restriction sites, which wereused for cloning the 3HR fragment downstream of the DHFR selectioncassette in the plasmid pT230 5HR-NcMic3-DHFR (XbaI at 5′ and NotI at 3′of the PCR fragment). The sequences of the primers are shown in Table Ibelow.

TABLE I List of the primers used for integration of the  5′UTR and 3′UTR sequences of the ncmic3 gene. Name of No. of the primer5′ → 3′ Sequence sequence 5 HR NCmic3  CGCGGTACCCATGTGAATATGCTTTA SEQ IDF KpnI ACCGTGAC NO: 1 5 HR NCmic3  CGCATCGATGAGCTATAACCCTTGGA SEQ IDR ClaI AATGACTC NO: 2 3 HR NCmic3  CGCTCTAGACATGCTGATGAAGAAGG SEQ IDF XbaI GAAGT NO: 3 3 HR NCmic3  CGCGCGGCCGCTCTCTCCTGAAGTCT SEQ ID R NotITCGAGACC NO: 4 The sequences of the restriction sites are underlined.

b) Conditions for Electroporation and Selection

50 μg of the plasmid pNcMic3KO-DHFR, purified and then linearized byNotI, was added to 5×10⁷ NC1 tachyzoites of Neospora caninum suspendedin the CYTOMIX electroporation medium containing ATP (3 mM) andglutathione (3 mM) (Van den Hoff et al., Nucleic Acid Research, June 11;20(11): 2902), and electroporation was carried out in a cuvette with a 4mm gap, in a volume of 800 μL on a BioRad apparatus (parameters: 2000 V,50 ohms, 25 μF, with two electric shocks).

After electroporation, the tachyzoites were deposited on a monolayer ofHFF cells in culture. For selection of the mutants, the culture mediumis replaced and supplemented with the selection agent (2 μMpyrimethamine), 24 h after electroporation. Three culture passages arecarried out in this medium.

After 16 days of selection, the resistant parasites are cloned by limitdilution in the wells of 96-well plates of HFF cells. Afteramplification, the lysis plaques caused by the parasite areinvestigated. The parasites are subcultured and their genomic DNA isextracted for PCR analyses. These PCR analyses should confirmintegration of the transgene but should also allow differentiation ofthe parasites that have randomly integrated the transgene from theparasites of interest the ncmic3 gene of which has been effectivelysuppressed by homologous recombination.

c) PCR Analysis

Starting from the genomic DNA, PCRs were carried out for:

-   -   investigating the size of the DNA fragment amplified with a set        of PCR primers No. 1: HR NCmic3 F (SEQ ID NO: 5) and HR NCmic3 R        (SEQ ID NO: 6), present on the homologous sequences. With random        integration of the transgene, two DNA fragments of 2163 bp and        of 3824 bp are amplified, whereas with homologous recombination,        only a fragment of 3824 bp is amplified. With the wild-type        strains, only a fragment of 2163 bp is amplified.    -   verifying the presence/absence of the ncmic3 gene with the set        of PCR primers No. 2: ORF NCmic3 F (SEQ ID NO: 7) and ORF NCmic3        R (SEQ ID NO: 8).    -   and/or verifying the presence/absence of the DHFR cassette with        the set of PCR primers No. 3: ORF DHFR F (SEQ ID NO: 9) and ORF        DHFR R (SEQ ID NO: 10).

The sequences of the primers and the size of the amplicons resultingfrom the different PCRs are shown in Table II and Table III below,respectively.

TABLE II List of the primers used for the different PCRsfor validation of the construction of the mutant strain Neo ncmic3 KO.No. Name of the No. of of primer 5′ → 3′ Sequence sequence PCRHR NCmic3 F GTCATCGACCGCCGGAACTA SEQ ID 1 GTAGT NO: 5 HR NCmic3 RGCAGAGGTTCTGCGTATCTA SEQ ID 1 ACACGG NO: 6 ORF NCmic3 FTTTCCCTTCTAAACACAGTCG SEQ ID 2 NO: 7 ORF NCmic3 R CCTTCAGTGGTTCTCCATGASEQ ID 2 GT NO: 8 ORF DHFR F CCTTCTCAGACAACGGGGTA SEQ ID 3 NO: 9ORF DHFR R AGATCTTCACGCCCTTCTCA SEQ ID 3 NO: 10 Integ NCmic3 FGAAAGTGTCAGTGGTAGAGAC SEQ ID 4 TGC NO: 11 and 6 ORF NCmic3 R2CCTTCACTCGAGATCGCGCAA SEQ ID 4 ATGAGC NO: 12 ORF DHFR R2GGACCTCTGTACGAGACATGC SEQ ID 6 CG NO: 13 Integ NCmic3 RTGTTTACAGGTGATCCAGAAA SEQ ID 5 AGG NO: 14 and 7 ORF NCmic3 F2GAATTTTGGGACAGGGGAAT SEQ ID 5 NO: 15 ORF DHFR F2 GTCTCTCGTTTTCCTCTCTTTSEQ ID 7 TCGG NO: 16

TABLE III Size of the amplicons (in base pairs) of the different PCRsfor validation of the construction of the mutant strain Neo ncmic3 KO.No. of Neospora PCR Neo ncmic3 KO caninum (NC1) 1 3824 2163 2 —  850 3 504 — 4 — 3127 5 — 3374 6 2890 — 7 3258 —

The electrophoretic profiles of the PCR products are presented in FIG.3-A. Among the clones studied, certain clones display a specific band ofDHFR (PCR 3) but no specific band of ncmic3 (PCR 2). PCR No. 1, whichwas carried out on these clones, revealed a band of 3824 bp specific fora Neo ncmic3 KO clone.

New PCR analyses were carried out on these clones of interest with newsets of primers. These PCRs, called integration PCRs, allow validationof the genetic KO using a primer present on the genome upstream ordownstream of the sequences flanking the ncmic3 gene and a second primerpresent in the selection cassette (dhfr gene) or in the gene of interest(ncmic3) (FIG. 3-B).

In FIG. 3-B, PCRs No. 4 and No. 5 make it possible to show the presenceof ncmic3 at the locus of ncmic3. PCR No. 4 is carried out with theprimer set Integ NCmic3 F (SEQ ID NO: 11) and ORF NCmic3 R2 (SEQ ID NO:12). PCR No. 5 is carried out with the primer set Integ NCmic3 R (SEQ IDNO: 14) and ORF NCmic3 F2 (SEQ ID NO: 15). The presence of bands for thewild-type strain NC1 of Neospora caninum and the absence of these bandsfor the mutant strain Neo ncmic3 KO are observed. In FIG. 3-B, PCRs No.6 and No. 7 make it possible to show the presence of DHFR at the locusof ncmic3. PCR No. 6 is carried out with the primer set Integ NCmic3 F(SEQ ID NO: 11) and ORF DHFR R2 (SEQ ID NO: 13). PCR No. 7 is carriedout with the primer set Integ NCmic3 R (SEQ ID NO: 14) and ORF DHFR F2(SEQ ID NO: 16). The absence of bands for the wild-type strain NC1 ofNeospora caninum and the presence of bands for the strain Neo ncmic3 KOare noted. The presence of a non-specific band for PCR No. 6 atapproximately 1000 bp should be noted.

All of the PCR results demonstrate that homologous recombination hasindeed taken place and that ncmic3 gene has indeed been deleted from themutant strain Neo ncmic3 KO.

d) Analysis by Immunofluorescence

Analysis was carried out by immunofluorescence. 24 h beforeimmunofluorescence analysis, 5×10⁵ parasites were deposited in a p24well containing a coverslip covered with a HFF cell lawn.

The cells infected by the parasites are washed twice with 1×PBS and thenfixed with paraformaldehyde (3.7% in 1×PBS) for 30 min After 3 washingswith 1×PBS, the cells are permeabilized with Triton solution (0.1% in1×PBS) for 5 minutes. After 3 washings with 1×PBS, a saturation step iscarried out with a solution of 1×PBS/10% FCS (foetal calf serum) for 30min. The cells are then incubated with the primary antibody diluted in asolution of PBS/2% FCS (foetal calf serum) for 1 hour, washed 3 timesand then incubated with the secondary antibody diluted in a solution ofPBS/2% FCS (foetal calf serum) for 1 hour. After 2 washings with 1×PBS,the coverslips are mounted on a slide with Immu-Mount and are observedwith a fluorescence microscope.

The primary antibody used is an antibody that allows detection ofexpression of the protein NcMIC3 in the parasite (primary antibody:rabbit anti-mic3 antibody and commercial secondary antibody: Alexafluor® 594 goat anti-rabbit, Life Technologies ref. A-11012).

For the wild-type strain NC1 of Neospora caninum, red fluorescence isobserved at the apical pole of the parasite, revealing the presence ofthe protein NcMIC3 (FIG. 4A), whereas for the mutant strain Neo ncmic3KO, no fluorescence is observed at the apical pole of the parasite,demonstrating absence of the protein NcMIC3 (FIG. 4B).

Example 2 Construction of the Mutant Strain Neo Ncmic1 KO

a) Construction of the Plasmid pNc mic1KO-CAT-GFP

The plasmid pNcMic1KO-CAT-GFP (FIG. 2-B) contains a CAT-GFP selectioncassette coding for a fusion protein giving both resistance tochloramphenicol (CAT) and green fluorescence (GFP: Green FluorescentProtein). The latter is placed under the control of the α-tubulinpromoter of Toxoplasma gondii to allow expression of the gene in theparasite. Either side of the cassette, the homologous regions of thesequences flanking the ncmic1 gene have been cloned.

The 3′ UTR region of the ncmic1 gene was amplified by PCR from thegenomic DNA of the strain NC1 of Neospora caninum. For theamplification, the primers 3 HR NCmic1 F KpnI and 3 HR NCmic1 R HindIII(SEQ ID NO: 17 and SEQ ID NO: 18) allow amplification of the 3′UTRregion of the ncmic1 gene and creation of two restriction sites, whichwere used for cloning the 3HR fragment upstream of the CAT-GFP selectioncassette into the plasmid pT230 CAT-GFP (KpnI at 5′ and HindIII at 3′ ofthe PCR fragment).

The 5′ UTR region of the ncmic1 gene was amplified by PCR from thegenomic DNA of the strain NC1 of Neospora caninum. For theamplification, the primers 5 HR NCmic1 F BamHI and 5 HR NCmic1 R NotI(SEQ ID NO: 19 and SEQ ID NO: 20) allow amplification of the 5′ UTRregion of the ncmic1 gene and creation of two restriction sites, whichwere used for cloning the 5HR fragment downstream of the CAT-GFPselection cassette into the plasmid pT230 3HRNcMic1CAT-GFP (BamHI at 5′and NotI at 3′ of the PCR fragment). The sequences of the primers areshown in Table IV below.

TABLE IV List of the primers used for integration of the5'UTR and 3'UTR sequences of the ncmic 1 gene. Name of No. of the primer5′ → 3′ Sequence sequence 3 HR NCmic1 CGCGGTACCAGGCAGAAGTAAAGA SEQ IDF KpnI AGGTTCCTC NO: 17 3 HR NCmic1  CGCAAGCTTTGATCACGCAAGAAA SEQ IDR HindIII AGAAGC NO: 18 5 HR NCmic1 CGCGGATCCCATTTGTAGATACGGT SEQ IDF BamHI TGCACAC NO: 19 5 HR NCmic1 CGCGCGGCCGCACATTCAGACGGC SEQ IDR NotI AGAACTCTG NO: 20 The sequences of the restriction sites areunderlined.

b) Conditions for Electroporation and Selection

50 μg of the plasmid pNcMic1KO-CAT-GFP, purified and then linearized byKpnI, must be added to 5×10⁷ NC1 tachyzoites suspended in CYTOMIXelectroporation medium containing ATP (3 mM) and glutathione (3 mM) (Vanden Hoff et al., Nucleic Acid Research, June 11; 20(11): 2902), andelectroporation must be carried out in a cuvette with a 4 mm gap, in avolume of 800 μL on a BioRad apparatus (parameters: 2000 V, 50 ohms, 25μF, with two electric shocks).

After electroporation, the tachyzoites will be deposited on a monolayerof HFF cells in culture. For selection of the mutants, the culturemedium will be replaced and supplemented with the selection agent (50 μMchloramphenicol), 24 h after electroporation. Three culture passagesmust be carried out in this medium.

After 15 days of selection, the resistant parasites will be cloned bylimiting dilution in the wells of 96-well plates of HFF cells. Afteramplification, the lysis plaques caused by the parasite will beinvestigated. The parasites will be subcultured and their genomic DNAwill be extracted for PCR analyses.

c) PCR Analysis

The sequences of the primers and the expected size of the ampliconsresulting from the different PCRs are shown in Table V and Table VIbelow, respectively.

TABLE V List of the primers used for the different PCRsfor validation of the construction of the mutant strains Neo ncmic1 KO.Name of the No. of No. of primer 5′ → 3′ Sequence sequence  PCRInteg NCmic1 F CCGAGCAAGTTAGCAAGTCC SEQ ID 1 and 3 NO: 21 ORF CATGFP RCCGTTTGGTGGATGTCTTCT SEQ ID 1 NO: 22 ORF CATGFP F GCATCGACTTCAAGGAGGACSEQ ID 2 NO: 23 Integ NCmic1 R CTTGTCCGTCACATCGTTTG SEQ ID 2 and 4NO: 24 ORF NCmic1 R TTCTCCAGGCACTCACCTCT SEQ ID 3 NO: 25 ORF NCmic1 FAGCTTCCAACAACGAGAGGA SEQ ID 4 NO: 26 ORF NCmic1 F2 CCCAGGATATCGTTTGTTGCSEQ ID 5 NO: 27 ORF NCmic1 R2 CTTCTGATGCACGGAACTGA SEQ ID 5 NO: 28ORF CATGFP F2 CCTGAAGTTCATCTGCACCA SEQ ID 6 NO: 29 ORFCATGFP R2GTAGTGGTTGTCGGGCAGCA SEQ ID 6 NO: 30

TABLE VI Size of the amplicons (in base pairs) of the different PCRs forvalidation of the construction of the mutant strain Neo ncmic1 KO. No.of Neospora PCR Neo ncmic1 KO caninum (NC1) 1 3359 — 2 3421 — 3 — 3746 4— 3046 5 —  449 6  472 —

Example 3 Construction of the Mutant Strain Neo Ncmic1-3 KO

a) Construction of the Plasmid pNc mic1KO-CAT-GFP Construction of theplasmid pNcMic1KO-CAT-GFP is described in Example 2 (2a).

b) Conditions for Electroporation and Selection

50 μg of the plasmid pNcMic1KO-CAT-GFP, purified and then linearized byKpnI, was added to 5×10⁷ Neo ncmic3 KO tachyzoites suspended in theCYTOMIX electroporation medium containing ATP (3 mM) and glutathione (3mM) (Van den Hoff et al., Nucleic Acid Research, June 11; 20(11): 2902),and electroporation was carried out in a cuvette with a 4 mm gap, in avolume of 800 μL on a BioRad apparatus (parameters: 2000 V, 50 ohms, 25μF, with two electric shocks).

After electroporation, the tachyzoites were deposited on a monolayer ofHFF cells in culture. For selection of the mutants, the culture mediumis replaced and supplemented with the selection agent (chloramphenicol50 μM), 24 h after electroporation. Three culture passages are carriedout in this medium.

After 15 days of selection, the resistant parasites are cloned bylimiting dilution in the wells of 96-well plates of HFF cells. Afteramplification, the lysis plaques caused by the parasite areinvestigated. The parasites are subcultured and their genomic DNA isextracted for PCR analyses.

c) PCR Analysis

The sequences of the primers and the size of the amplicons resultingfrom the different PCRs are shown in Table VII and Table VIII below,respectively.

TABLE VII List of the primers used for the different PCRsfor validation of the construction of the mutantstrains Neo ncmic3 KO and Neo ncmic1-3 KO. No. Name of the No. of ofprimer 5' → 3' Sequence sequence PCR Integ NCmic1 F CCGAGCAAGTTAGCAAGTCCSEQ ID 1 NO: 21 and 3 ORF CATGFP R CCGTTTGGTGGATGTCTTCT SEQ ID 1 NO: 22ORF CATGFP F GCATCGACTTCAAGGAGGAC SEQ ID 2 NO: 23 Integ NCmic1 RCTTGTCCGTCACATCGTTTG SEQ ID 2 NO: 24 and 4 ORF NCmic1 RTTCTCCAGGCACTCACCTCT SEQ ID 3 NO: 25 ORF NCmic1 F AGCTTCCAACAACGAGAGGASEQ ID 4 NO: 26 Integ NCmic3 F GAAAGTGTCAGTGGTAGAGAC SEQ ID 5 TGC NO: 11and 7 ORF NCmic3 R2 CCTTCACTCGAGATCGCGCAA SEQ ID 5 ATGAGC NO: 12ORF DHFR R2 GGACCTCTGTACGAGACATGC SEQ ID 7 CG NO: 13 Integ NCmic3 RTGTTTACAGGTGATCCAGAAA SEQ ID 6 AGG NO: 14 and 8 ORF NCmic3 F2GAATTTTGGGACAGGGGAAT SEQ ID 6 NO: 15 ORF DHFR F2 GTCTCTCGTTTTCCTCTCTTTSEQ ID 8 TCGG NO: 16 ORF NCmic1 F2 CCCAGGATATCGTTTGTTGC SEQ ID 9 NO: 27ORF NCmic1 R2 CTTCTGATGCACGGAACTGA SEQ ID 9 NO: 28 ORF CATGFP F2CCTGAAGTTCATCTGCACCA SEQ ID NO: 29 10 ORFCATGFP R2 GTAGTGGTTGTCGGGCAGCASEQ ID 10 NO: 30 ORF NCmic3 F TTTCCCTTCTAAACACAGTCG SEQ ID 11 NO: 7ORF NCmic3 R CCTTCAGTGGTTCTCCATGA SEQ ID 11 GT NO: 8 ORF DHFR FCCTTCTCAGACAACGGGGTA SEQ ID 12 NO: 9 ORF DHFR R AGATCTTCACGCCCTTCTCASEQ ID 12 NO: 10

TABLE VIII Size of the amplicons (in base pairs) of the different PCRsfor validation of the construction of the mutant strains Neo ncmic3 KOand Neo ncmic1-3 KO. No. of Neospora PCR Neo ncmic1-3 KO caninum (NC1)Neo ncmic3 KO 1 3359 — — 2 3421 — — 3 — 3746 3746 4 — 3046 3046 5 — 3127— 6 — 3374 — 7 2890 — 2890 8 3258 — 3258 9 —  449  449 10  472 — — 11 — 850 — 12  504 —  504

In FIG. 5, PCR No. 1 is carried out with the set of primers Integ NCmic1F (SEQ ID NO: 21) and ORF CATGFP R (SEQ ID NO: 22). PCR 2 is carried outwith the set of primers ORF CATGFP F (SEQ ID NO: 23) and Integ NCmic1 R(SEQ ID NO: 24). PCR No. 3 is carried out with the set of primers IntegNCmic1 F (SEQ ID NO: 21) and ORF NCmic1 R (SEQ ID NO: 25). PCR No. 4 iscarried out with the set of primers Integ NCmic1 R (SEQ ID NO: 24) andORF NCmic1 F (SEQ ID NO: 26). PCR No. 5 is carried out with the set ofprimers Integ NCmic3 F (SEQ ID NO: 11) and ORF NCmic3 R2 (SEQ ID NO:12). PCR No. 6 is carried out with the set of primers Integ NCmic3 R(SEQ ID NO: 14) and ORF NCmic3 F2 (SEQ ID NO: 15). PCR No. 7 is carriedout with the set of primers Integ NCmic3 F (SEQ ID NO: 11) and ORF DHFRR2 (SEQ ID NO: 13). PCR No. 8 is carried out with the set of primersInteg NCmic3 R (SEQ ID NO: 14) and ORF DHFR F2 (SEQ ID NO: 16). PCR No.9 is carried out with the set of primers ORF NCmic1 F2 (SEQ ID NO: 27)and ORF NCmic1 R2 (SEQ ID NO: 28). PCR No. 10 is carried out with theset of primers ORF CATGFP F2 (SEQ ID NO: 29) and ORF CATGFP R2 (SEQ IDNO: 30). PCR No. 11 is carried out with the set of primers ORF NCmic3 F(SEQ ID NO: 7) and ORF NCmic3 R (SEQ ID NO: 8). PCR No. 12 is carriedout with the set of primers ORF DHFR F (SEQ ID NO: 9) and ORF DHFR R(SEQ ID NO: 10).

The electrophoretic analyses of the PCR products show that the strainNeo ncmic1-3 KO no longer has the ncmic1 and ncmic3 genes (wells 3, 4,5, 6, 9 and 11, FIG. 5) and does have the dhfr and cat-gfp genes (wells1, 2, 7, 8, 10 and 12, FIG. 5), thus validating production of the strainNeo ncmic1-3 KO. All of the PCR results demonstrate that homologousrecombination has indeed taken place and the strain Neo ncmic1-3 KO hasindeed been deleted from the ncmic1 and ncmic3 genes.

d) Immunofluorescence Analysis

Immunofluorescence analysis was carried out solely by direct observationof the fluorescence of the parasite (FIG. 6).

The parasites of the two mutant strains are visualized in direct light(images A and C). One and the same microscopic field is visualized influorescence. Green fluorescence, due to expression of the recombinantchimeric protein CAT-GFP, is only detected in the mutant strain Neoncmic1-3 KO (image D) following insertion of the CAT-GFP cassette.Conversely, the strain Neo ncmic3 KO, which does not have a CAT-GFPcassette, does not express the CAT-GFP protein and consequently does notdisplay fluorescence (image B).

Example 4 Immunostimulation of the Mouse Pups with the Mutant ToxoMic1-3 KO 1-Experimental Protocol

1.1-Animals

Immunostimulation is carried out on the C57BL/6 mouse pups aged 3 days.These mouse pups were obtained and bred in the INRA Centre in Nouzilly(Indre et Loire, France). The mouse pups are kept throughout theexperiments in an animal house of containment level 2 in order tominimize the risk of external contamination.

1.2-Strain T. gondii

1.2.1-Strain Toxo mic1-3 KO

The mutant strain of Toxoplasma gondii, in which the genes coding forthe proteins MIC1 and MIC3 were knocked out (called strain Toxo mic1-3KO) is maintained by successive passages on a human foreskin fibroblast(HFF) line cultured in DMEM medium supplemented with 10% of foetal calfserum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 μg/mL ofstreptomycin.

1.2.2-Strain RH

The wild-type strain RH of Toxoplasma gondii, from which the strain Toxomic1-3 KO is derived, is also maintained by successive passages on ahuman foreskin fibroblast (HFF) line cultured in DMEM mediumsupplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50U/mL of penicillin and 50 μg/mL of streptomycin.

For preparation of the total parasite extract, the tachyzoites of thestrain RH are washed, sonicated at 60 W/s, three times for 10 min andcentrifuged at 2000 g for 30 min at 4° C. The supernatant isconcentrated and divided into aliquots. The concentration is determinedby BCA assay, using BSA (Bovine Serum Albumin) as standard. The aliquotsare stored at −20° C.

1.3-Immunostimulation

Batches of 3-day-old C57BL/6 mouse pups were treated as follows:

6 mouse pups (batch A) served as unvaccinated control batch,

6 mouse pups (batch B) received the mutant Toxo mic1-3 KO

On D0, the mouse pups in batch A received 20 tachyzoites of the strainToxo mic1-3 KO by intraperitoneal route.

On D3 post-immunostimulation, 3 mouse pups from each batch weresacrificed for investigating the humoral immune response, theinflammatory response and parasitaemia.

On D9 post-immunostimulation, the 3 remaining mouse pups of each batchwere sacrificed for investigating the humoral immune response, theinflammatory response and parasitaemia.

1.4-Humoral Immune Response

The humoral immune response was studied by evaluating, by ELISA, thekinetics of appearance of the IgM specific anti-Toxoplasma gondiiantibodies in the serum. The sera are taken at the moment of sacrificeof the mouse pups on D3 and on D9 post-infection. The sample is left for10 mM at room temperature to allow clotting. The serum is recovered bycentrifuging the samples at 2000 rpm for 10 min at +20° C. Thesupernatant is divided into aliquots in a clean tube and stored at −20°C.

The total extract of the RH strain of Toxoplasma gondii, obtained asdescribed above, is used for sensitizing the flat-bottomed wells ofmicrotitre plates (Nunc). 100 μL of extract (at a concentration of 10μg/mL in carbonate buffer at 50 mM and pH=9.6) is deposited in eachwell. After one night at +4° C., three washings are carried out in PBSbuffer with the addition of 0.05% Tween-20 (PBS-T).

The non-specific sites are saturated by incubation of the plates for 1.5h at 37° C. under humid atmosphere with PBS with 4% BSA.

100 μL of each sample of serum diluted in PBS-T (1/50 dilution) isdeposited and incubated for 1 h at 37° C. under humid atmosphere.

After two series of three washings, 100 μL of anti-mouse IgM coupled toalkaline phosphatase (ALP; Sigma), diluted to 1/5000 in PBS-T, isdeposited and incubated for 1.5 h at 37° C. under humid atmosphere. Twonew series of three washings are carried out. Development is carried outusing 100 μL of paranitrophenylphosphate (PNPP) at 1 mg/mL in DEA-HCL.

Reading is carried out after incubation for 10 to 20 minutes, on a platereader (Wallac 1420 Multilabel counter) at a wavelength λ=405 nm.

The assumed positivity threshold was determined as a function of thevalues of absorbance (OD) of the mouse pups in the control batch: it isfixed at 0.23 of OD for a 1/50 serum dilution.

1.5-Investigation of the Inflammatory Response

Analysis of the inflammatory response of the mouse pups at intestinallevel was quantified by quantitative PCR (qPCR) by amplification of thegenes coding for IL-12 and IFN-γ. IL-12 is a cytokine produced inresponse to intrusion of a pathogen that stimulates secretion of IFN-γ,a cytokine produced by the immune system cells in response toinflammation on the site of infection by the pathogen.

After sacrifice of the mouse pups on D3 and D9, the intestine is removedand the ileum (1 cm above the caecum) is used for analysis by qPCR. Thetissue is incubated in 1 mL of Trizol® (Invitrogen) and then is groundin a Thurax, incubated at room temperature for 5 min and centrifuged for10 minutes at 12,000 g. The supernatant is then recovered and mixed bypipetting up and down approximately ten times.

Once the nuclear protein complexes have been dissociated with Trizol®,the RNA is isolated from the DNA and from the proteins with chloroform.One millilitre of chloroform is added to the supernatant and theTrizol®/chloroform mixture is stirred vigorously for 15 seconds, thenincubated at room temperature and finally centrifuged at 12,000 g for 15minutes at 4° C. After centrifugation, the mixture is separated into anorganic phase (phenol/chloroform, pink, lower phase), an interphase(white film, DNA and cell debris) and an aqueous phase (upper phase)containing the total RNAs.

The aqueous phase containing the total RNAs is recovered and 500 μL ofisopropanol is added to precipitate the RNA. The solution is stirred,incubated at room temperature for 10 min and centrifuged at 12,000 g for10 min at 4° C. The pellet obtained is isolated and then washed with 1mL of 75% ethanol (absolute ethanol diluted in 0.1% DEPC water andstored at −20° C.), stirred and centrifuged at 7500 g at 4° C. for 10min. The pellet is dried for 10 min under a fume hood in ice. Finallythe RNA is taken up in approximately 20 μL of 0.1% DEPC water.

The quality of extraction of the RNA is verified by electrophoresis on1% agarose gel and by calculating the direct ratio of the absorbance ata wavelength of 260 nm to that at a wavelength of 280 nm. This ratiomust be close to 2. Finally, the RNA extraction yield is quantifiedusing a spectrophotometer.

Two micrograms of RNA are incubated with 1 μL of Oligo dT (Eurogentec133 pmol/μl) in a final volume of 11 μL at 65° C. for 10 min, and then 2min in ice. Once the oligonucleotides dT are fixed on the polyA tail,the RNA solution is incubated with 2 μL of dNTP (dATP, dTTP, dGTP, dCTPeach at 20 mM), 4 μL of reverse transcriptase buffer (5× Eurogentec; 250mM Tris-HCl (pH 8.3); 375 mM KCl; 50 mM DTT; 15 mM MgCl₂) and 0.4 μL ofMuMLV (25 U/μ L; 50 mM Tric-HCL (pH 8.3); 1 mM EDTA, 0.1% Triton X-100,0.1 M NaCl; 5 mM DTT; 50% (v/v) glycerol) in a final volume of 20 μL for1.5 h at 37° C. The reverse transcriptase is then inhibited at 85° C.for 10 min.

In the present case, expression of the genes coding for the proteinsIL-12 and IFN-γ, expressed during the inflammatory response, wasquantified.

For amplification, the primer pair SEQ ID NO: 31(5′-CTCACATCTGCTGCTCCACAA-3′) and SEQ ID NO: 32(5′-GACGCCATTCCACATGTCACT-3′) was used for IL-12, the primer pair SEQ IDNO: (5′-TCTTCTTGGATATCTGGAGGAA-3′) and SEQ ID NO: 34(5′-AGCTCATTGAATGCTTGGCGCTG-3′) was used for assaying IFNγ and theprimer pair SEQ ID NO: 35 (5′-GGATACAGGCCAGACTTTGTTG-3′) and SEQ ID NO:36 (5′-GAGGGTAGGCTGGCCTATAG-3′) was used for assaying the murine HPRTreference gene.

Two microlitres of cDNA diluted to 1/10 from the reverse transcriptionreaction is incubated with 0.3 μL of the 5′ primer (25 μM); 0.3 μL ofthe 3′ primer (25 μM); 7.5 μL of Mix PCR (BioRad) in a final volume of15 μL. The conditions selected for the PCR reaction are as follows: 1)denaturation at 95° C. for 5 minutes, 2) denaturation at 95° C. for 10seconds, 3) pairing and elongation at 62° C. for IL-12, IFN′ and HPRTfor 15 seconds, 4) repeating the cycle starting from step 2: 39 times,5) melting curve from 55° C. to 95° C. to verify the presence or absenceof the dimers.

1.6-Investigation of the State of Infection of the Mouse Pups

The level of infection of the mouse pups was analysed by three differenttechniques: 1) tissue dissemination of the tachyzoites on HFF cells, 2)immunohistology on sections of intestine from the infected mouse pupsand 3) PCR from the ileum of the mouse pups in batches A and B.

1.6.1-Tissue Dissemination of the Tachyzoites on HFF Cells

The HFF cells are deposited in a 24-well plate one week beforedepositing the organs at a rate of 1×10⁴ cells/well. The cells arecultured in 1 mL of DMEM cell culture medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin.

After sacrifice, the spleen of the mouse pups of batches 1 and 2 isremoved and ground in 2 mL of 1×PBS. Ten microlitres of ground materialis deposited per well containing the HFF cells. Twenty-four hours afterdepositing the ground materials from the spleen, the cells are washedwith DMEM and then 1 mL of clean medium is deposited in each well. Thecells are incubated at 37° C., 5% CO₂ until the lysis plaques revealingthe presence of Toxo mic1-3 KO tachyzoites are detected.

1.6.2-Immunohistology on Sections of Intestine from the Infected MousePups

The intestine of the sacrificed mouse pups is rolled up like a Swissroll and then kept in this form using paper wrapped around the Swissroll and stapled. The samples are fixed in 4% paraformaldhehydesolution, diluted in 1×PBS (pH=7) and incubated for 8 hours at 4° C. Thetissues are then washed in 1×PBS and then incubated at 4° C. for 8 h inclean 1×PBS. This last step is repeated a second time. The tissues areincubated for 8 hours at 4° C., in 30% sucrose solution diluted in 1×PBSand filtered. Finally the tissues are transferred to moulds of asuitable size filled with OCT embedding medium. After 5 minutes ofincubation in the OCT, the samples are frozen using dry ice and storedat −80° C.

The samples of intestine are then cut into sections using a cryostatwhich maintains the sample at −20° C. Histological sections with athickness of 7 μm are prepared and then deposited on a slide byelectrostatic force. The slides are stored at −80° C.

The slides of histological sections are thawed and then left to dry atroom temperature for 1 h. The zone of the sample on the slide isdelimited with a pen with hydrophobic ink (Dakocitamation pen). Thehistological sections of intestine are permeabilized with 50 μL of asolution of 1×PBS, Mg²⁺, Ca²⁺ free, Triton X-100 and 1% BSA at roomtemperature and in a humid chamber for 10 min. The permeabilizingsolution is removed by aspiration and the samples are saturated in 50 μLof a solution of 1×PBS, Mg²⁺, Ca²⁺ free, Triton X-100 and 10% BSA atroom temperature and in a humid chamber for 1 h. After aspiration of thesaturation solution, 50 μL of rabbit anti-SAG1 polyclonal serum ofToxoplasma gondii diluted to 1/100 in a solution of 1×PBS, Mg²⁺, Ca²⁺free, Triton X-100 and BSA 1%, is deposited per sample. The samples areincubated at 4° C., in a humid chamber for 8 h. Two washings for5-minutes in 1×PBS, Mg²⁺, Ca²⁺ free, Triton X-100 and 1% BSA are carriedout. 50 μL of swine anti-rabbit antibodies coupled to fluoresceinisothiocyanate diluted to 1/20 in a solution of 1×, Mg²⁺, Ca²⁺ free,Triton X-100 and 1% BSA is deposited per sample. The samples areincubated in the presence of the coupled secondary antibody, at roomtemperature, in a humid chamber for 1 h 20 min Two washings for 5-minutein 1×PBS, Mg²⁺, Ca²⁺ free, Triton X-100 and 1% BSA are carried out andthen a final washing of the samples is carried out in sterile distilledwater. The slides are dried and then a drop of fluoromount G isdeposited on each sample. Finally, the samples are mounted between theslide and the cover slip.

1.6.3-Detection of SAG-1

A PCR was carried out from cDNAs obtained by reverse transcriptionreaction (cf. paragraph 1.5). The SAG-1 gene specific to the parasiteToxoplasma gondii was amplified by PCR with the primers SEQ ID NO: 37(5′-CTGCACCACTTCATTATTTCTTCTG-3′) and SEQ ID NO: 38(5′-ACTCACGCGACACAAGCTG-3′).

2 μL of cDNA is incubated with 1 μL of 5′ primer (10 μM); 1 μL of 3′primer (10 μM); 25 μL of GoTaq® Green Master Mix (2×, Promega) in afinal volume of 50 μL. The conditions selected for the PCR are asfollows: 1) denaturation at 94° C. for 5 min, 2) denaturation at 94° C.for 30 s, 3) pairing at 60° C. for 30 s, 4) elongation at 72° C. for 1min, 5) repeating the cycle starting from step 2: 34 times, 6)elongation at 72° C. for 5 min. The presence of the parasite Toxoplasmagondii in the intestine is verified by an amplified fragment of 1001base pairs.

2-Results

2.1-Investigation of the Humoral Response Post-Immunostimulation

The results of the ELISA assays on D3 and D9 post-infection for the seraof the batches of mouse pups, controls and immunostimulated with thestrain Toxo mic1-3 KO, are shown in FIG. 7. The mouse pups in thecontrol batch (A) have not developed a humoral response on D3 and on D9post-infection in contrast to 2 mouse pups out of 3 of the batchimmunostimulated with the strain Toxo mic1-3 KO (B), which producedanti-toxoplasmic IgM antibodies at 9 days post-infection; these are themouse pups No. 4 and No. 6.

2.2-Investigation of the Inflammatory Response Post-Immunostimulation

The results of the quantitative PCR tests for expression of the genecoding for IFN-γ and the gene coding for IL-12 carried out on D9post-immunostimulation are shown in FIGS. 8-A and 8-B respectively. Themouse pups in the control batch (A) have not developed an inflammatoryresponse (secretion of IL-12 and IFN-γ) on D9 post-infection. Incontrast, 9 days after immunostimulation, the mouse pups No. 4, 5 and 6display significantly greater expression of IL-12 than that of the mousepups in the control batches (FIG. 8-B). Similarly, two mouse pups out of3 in the batch immunostimulated with the strain Toxo mic1-3 KO displayexpression of IFN-γ significantly greater than that of the mouse pups inthe control batches (FIG. 8-A); these are the mouse pups No. 4 and No.6.

2.3-Investigation of the state of infection of the mouse pups

2.3.1-Dissemination of the Spleens on HFF Cells

The dissemination of the spleens on HFF cells is illustrated in TableIX. No lysis plaque is detected in the HFF cells infected with thespleens from the mouse pups of the control batch. In contrast, the HFFcells infected with the ground material from the spleen originating fromthe mouse pups immunostimulated with the strain Toxo mic1-3 KO havelysis plaques, demonstrating that tachyzoites are present in the spleen.

Moreover, the technique of tissue dissemination of the tachyzoites onHFF cells is a semiquantitative technique which shows that the mousepups in the batch immunostimulated with the strain Toxo mic1-3 KO havedifferent states of infection. The mouse pups No. 4 and No. 6 seem bethe most infected as they have the largest number of parasites in thespleen, followed by mouse pup No. 5, which has far fewer parasites inthe spleen (Table IX).

TABLE IX Result of optical microscopy analysis, of the dissemination ofthe organs on human fibroblast cells (HFF). The tachyzoites observedoriginate from the spleens of the mouse pups immunostimulated with thestrain Toxo mic1-3 KO and form lysis plaques when they colonize the HFFcells. Presence of Lysis plaques parasites observed Control mouse pup 4NO 0 Control mouse pup 5 NO 0 Control mouse pup 6 NO 0 ImmunostimulatedYES >70% of the lysed mouse pup 4 cells Immunostimulated YES 2 mouse pup5 Immunostimulated YES >70% of the lysed mouse pup 6 cells

2.3.2-Immunohistology

The immunohistology sections of the intestines from the mouse pups areillustrated in FIG. 9.

For the mouse pups in the control batch, no tachyzoite was observed onthe immunohistology sections.

The sections of intestine from the mouse pups No. 4 (B) and No. 6 (D)and No. 5 respectively (data not shown), immunolabelled with a rabbitanti-T. gondii polyclonal antibody, allow visualization of the presenceof tachyzoites of the strain Toxo mic1-3 KO (white dots) for the mousepup No. 4 only.

2.3.3-PCR

The PCR results are presented in FIG. 10. Amplification of the SAG1 geneof Toxoplasma gondii from a DNA sample originating from ground materialfrom the ileum is representative of the presence of Toxo mic1-3 KOtachyzoites (amplified fragment at 1001 bp). This semiquantitativetechnique shows absence of a band for the mouse pups in the controlbatch (NV) sacrificed on D3 (T1) or on D9 (T2).

For the mouse pups immunostimulated with the strain Toxo mic1-3 KO (V),no band corresponding to the SAG1 gene is detected 3 days afterinfection. On D9, two mouse pups out of 3 display a band of DNA of 1001base pairs, providing evidence of the presence of Toxo mic1-3 KOtachyzoites. The intensity of the bands observed between the mouse pupsNo. 4 and No. 6 is clearly different. As the intensity of the bands isproportional to the number of parasites present in the intestine, themouse pup No. 4 has more parasites in the intestine than the mouse pupNo. 6, 9 days post-infection. These results confirm the observation madeby immunohistology.

Example 5 Protection Against Cryptosporidiosis of the Mouse PupsImmunostimulated with the Mutant Toxo Mic1-3 KO by Intraperitoneal Route1-Experimental Protocol

1.1-Animals

Immunostimulation is carried out on C57BL/6 mouse pups aged 3 days.These mouse pups were obtained and bred in the INRA Centre in Nouzilly(Indre et Loire). The mouse pups are kept throughout the experiments inan animal house of containment level 2 in order to limit the risk ofexternal contamination as far as possible.

1.2-Cryptosporidium parvum

The oocysts of Cryptosporidium parvum are obtained from excrement ofcalves infected with 10⁷ C. parvum oocysts. The stool undergoes varioustreatments until a suspension of purified, sterile parasites isobtained, suitable for use in cell culture. Throughout the treatment,the oocysts are manipulated at 4° C., to prevent excystation of theoocysts.

Briefly, after recovery of the stool, the latter is diluted in freshwater and then passed through a 100-μm filter and centrifuged at 1900 gfor 10 minutes at 4° C. The pellets obtained, containing the oocysts,are taken up in 2% potassium dichromate solution (Prolabo, ref. 26 776290, CAS 7778-50-9), then washed twice with cold water by centrifugationat 1900 g for 10 minutes at 4° C. to remove the potassium dichromate.After washing, the pellet of coccidia is taken up in a mixture of waterand ether (Ethyl Ether, Carlo Erba, CAS No. 60-29-7) diluted to 1/5, andthen is centrifuged again at 1900 g for 10 minutes at 4° C. The upperphases containing the fats and the ether are removed and the pellet isrecovered and taken up in cold water after passing through a 20-μmfilter. Two to three millilitres of the suspension of parasites obtainedis deposited on a glucose gradient prepared from Sheather solution(sucrose 500 g, water 320 ml, 0.2 g of sodium azide (Prolabo, CAS26628-22-8)). Two rings of oocysts are formed after centrifugation ofthe glucose gradient at 2000 g for 20 minutes at 4° C. The two rings arerecovered and washed several times in cold water. The purified oocystsare then sterilized. After centrifugation at 1900 g for 10 minutes at 4°C., the pellet of oocysts is incubated for 15 minutes in a solution ofbleach (sodium hypochlorite (Sigma 239305-500ML Titre: 4.5% of activechlorine) diluted to 10% in demineralized water, then washed 3 times insterile 1×PBS (diluted from a solution of 10×PBS: sodium chloride, NaCl80 g/litre water; potassium chloride, KCl 2 g/litre; potassiumdihydrogen phosphate, KH₂PO₄: 2 g/litre; disodium hydrogen phosphate,Na₂HPO₄, 12 H₂O: 29 g/litre). The purified and sterilized oocysts arecounted on a slide (5 μL of solution of oocysts and 495 μL of malachitegreen), adjusted to a concentration of 2×10⁸ oocysts/mL, divided intoaliquots in 1.5-mL tubes and stored at 4° C.

1.3-Strain Toxo Mic1-3 KO

The mutant strain of Toxoplasma gondii, in which the genes coding forthe proteins MIC1 and MIC3 have been knocked out (called strain Toxomic1-3 KO) is maintained by successive passages on a human foreskinfibroblast (HFF) line cultured in DMEM medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin.

1.4-Immunostimulation

Batches of C57BL/6 mouse pups aged 3 days were treated as follows:

7 mouse pups (batch 1) received the mutant Toxo mic1-3 KO

7 mouse pups (batch 2) served as unvaccinated control batch.

On D0, the mouse pups in batch 1 received 20 tachyzoites of the strainToxo mic1-3 KO by intraperitoneal route.

On D3 post-immunostimulation, the mouse pups in batch 1 and batch 2 werechallenged with 500,000 parasites of Cryptosporidium parvum.

On D9 post-infection, the mouse pups in batch 1 and 2 were sacrificed inorder to evaluate protection against Cryptosporidium parvum.

1.5-Investigation of the State of Infection of the Mouse Pups

The state of infection of the mouse pups was analysed by the techniqueof tissue dissemination of the tachyzoites on HFF cells described above.The HFF cells are deposited in a 24-well plate, one week beforedepositing the organs at a rate of 10⁴ cells per well. The cells arecultured in 1 mL of DMEM cell culture medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin. After sacrifice of the mouse pups, the spleen ofeach of the mouse pups is taken and ground in 2 mL of 1×PBS. Tenmicrolitres of ground material is deposited per well containing the HFFcells cultured in 1 mL of medium. Twenty-four hours after depositing theground material from the spleen, the cells are washed in DMEM and then 1mL of clean medium is deposited per well. The cells are cultured untilthe lysis plaques revealing the presence of Toxo mic1-3 KO tachyzoitesare detected.

1.6-Investigation of Protection

The protection of the mouse pups against Cryptosporidium parvum isanalysed by counting the oocysts in the intestine. After the mouse pupsare sacrificed, the intestines are recovered, weighed, placed in 1 mL ofwater (4° C.) and ground for 20 seconds. 100 microlitres of groundmaterial is added to 400 μL of sugar solution at 4° C. (500 g ofpowdered sugar, 320 mL of distilled water, Na azide at 0.02%). Afterhomogenizing the solution by pipetting, 20 μL is deposited on a Thomacell. Before counting the oocysts, the slides are kept in a cool placefor 15 minutes to allow the oocysts in the sugar solution to rise to thesurface. The total number of oocysts in the intestine is calculated bymeans of the following formula: Total number of oocysts of C. parvum inthe intestine=Number of oocysts counted on Thoma slide×dilutionfactor×10 000×(1+weight of the mouse pup intestine).

2-Results

2.1-Investigation of the State of Infection of the Mouse Pups

The state of infection of the mouse pups is evaluated from parasitaemiain the case of Cryptosporidium parvum (C. parvum) and Toxoplasma gondii(T. gondii). Parasitaemia in the case of C. parvum is determined by thetotal number of oocysts of C. parvum counted in the mouse pup intestine.Parasitaemia in the case of T. gondii is evaluated by dissemination ofthe spleens of the mouse pups on HFF cells. The parasitaemias for C.parvum and T. gondii are presented in Table X.

TABLE X Summary table of parasitaemia observed in the case of C. parvumand in the case of T. gondii of the batch of the mouse pups infectedwith C. parvum (infected C. parvum) and of the batch of the mouse pupsinoculated with T. gondii and infected with C. parvum (inoculated T.gondii + C. parvum): t, tachyzoites; NR, data not reported. The numberassociated with parasitaemia in the case of T. gondii corresponds to thenumber of tachyzoites counted in 20 μL of supernatant of the HFF cellsinfected after dissemination of the spleen. Infected C. parvumInoculated T. gondii + C. parvum Total Total number of Parasit- numberof Parasit- mouse oocysts of aemia mouse oocysts of aemia pups C. parvumT. gondii pups C. parvum T. gondii 1 862500 NO 1 935000 SLIGHT (6.6 10⁵t.) 2 966500 NO 2 53000 YES (NR) 3 1083000 NO 3 55000 YES (2 10⁶ t.) 4565000 NO 4 1568000 VERY SLIGHT (2 10⁵ t.) 5 684000 NO 5 832500 VERYSLIGHT (1.2 10⁵ t.) 6 847500 NO 6 754000 VERY SLIGHT (1.6 10⁵ t.) 7994500 NO 7 330000 YES (1.24 10⁶ t.)

The mouse pups infected with C. parvum only, have a total number ofoocysts of C. parvum in the intestine in the range from 5×10⁵ to 1×10⁶oocysts.

The mouse pups immunostimulated with the strain Toxo mic1-3 KO and theninfected with C. parvum display T. gondii parasitaemia varying from onemouse pup to another. For mouse pups No. 2, 3 and 7, which have thehighest T. gondii parasitaemia, the total number of oocysts of C. parvumin the intestine varies from 5×10⁴ to 3.3×10⁵ oocysts. These resultsdemonstrate the correlation that exists between parasitaemia with Toxomic1-3 KO and the reduction in C. parvum infection.

2.2-Investigation of Protection

The state of protection of the mouse pups is shown in FIG. 11.

In the batch of the mouse pups vaccinated by intraperitoneal route withthe mutant Toxo mic1-3 KO, 3 mouse pups out of 7 have a total number ofoocysts of C. parvum in the intestine that is significantly reduced, byapproximately 62% to 94%. These three mouse pups are the mouse pups No.2, No. 3 and No. 7, which displayed the highest parasitaemia with T.gondii of all the mouse pups in this group.

Example 6 Protection Against Cryptosporidiosis, of the Mouse PupsImmunostimulated with the Mutant Toxo Mic1-3 KO by Oral Route1-Experimental Protocol

1.1-Animals

Immunostimulation is carried out on C57BL/6 mouse pups aged 3 days.These mouse pups were obtained and bred in the INRA Centre in Nouzilly(Indre et Loire). The mouse pups are kept throughout the experiments inan animal house of containment level 2 in order to limit the risk ofexternal contamination as far as possible.

1.2-Cryptosporidium parvum

Oocysts of Cryptosporidium parvum are obtained from excrement of calvesinfected with 10⁷ C. parvum oocysts. The stool undergoes varioustreatments until a suspension of sterile, purified parasites isobtained, suitable for use in cell culture. Throughout the treatment,the oocysts are manipulated at 4° C. to prevent excystation of theoocysts.

Briefly, after recovery of the stool, the latter is diluted in freshwater and then passed through a 100-μm filter and centrifuged at 1900 gfor 10 minutes at 4° C. The pellets obtained, containing the oocysts,are taken up in 2% potassium dichromate solution (Prolabo ref. 26 776290, CAS 7778-50-9), then washed twice with cold water by centrifugationat 1900 g for 10 minutes at 4° C. to remove the potassium dichromate.After washing, the pellet of coccidia is taken up in a mixture of waterand ether (ethyl ether, Carlo Erba CAS No. 60-29-7) diluted to 1/5, thencentrifuged again at 1900 g for 10 minutes at 4° C. The upper phasescontaining the fats and the ether are removed and the pellet isrecovered and taken up in cold water after passing through a 20-μmfilter. Two to three millilitres of the suspension of parasites obtainedis deposited on a glucose gradient prepared from Sheather solution(sucrose 500 g, water 320 ml, 0.2 g of sodium azide (Prolabo, CAS26628-22-8)). Two rings of oocysts are formed after centrifugation ofthe glucose gradient at 2000 g for 20 minutes at 4° C. The two rings arerecovered and washed several times in cold water. The purified oocystsare then sterilized. After centrifugation at 1900 g for 10 minutes at 4°C., the pellet of oocysts is incubated for 15 minutes in a solution ofbleach (sodium hypochlorite, Sigma 239305-500ML Titre: 4.5% of activechlorine) diluted to 10% in demineralized water and then washed 3 timesin sterile 1×PBS (diluted from a solution of 10×PBS: sodium chloride,NaCl 80 g/litre water; potassium chloride, KCl 2 g/litre; potassiumdihydrogen phosphate, KH₂PO₄: 2 g/litre; disodium hydrogen phosphate,Na₂HPO₄, 12 H₂O: 29 g/litre). The purified and sterilized oocysts arecounted on a slide (5 μL of solution of oocysts and 495 μL of malachitegreen), adjusted to a concentration of 2×10⁸ oocysts/mL, divided intoaliquots in 1.5-mL tubes and stored at 4° C.

1.3-Strain Toxo mic1-3 KO

The mutant strain of Toxoplasma gondii, in which the genes coding forthe proteins MIC1 and MIC3 have been suppressed (called strain Toxomic1-3 KO) is maintained by successive passages on a human foreskinfibroblast (HFF) line cultured in DMEM medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin.

1.4-Immunostimulation

Batches of C57BL6 mouse pups aged 3 days were treated as follows:

4 mouse pups (batch 1) received the mutant Toxo mic1-3 KO

4 mouse pups (batch 2) served as untreated control batch.

On D0, the mouse pups in batch 1 received 20,000 tachyzoites of thestrain Toxo mic1-3 KO by oral route.

On D3 post-immunostimulation, the mouse pups in batch 1 and batch 2 werechallenged with 1,000,000 parasites of Cryptosporidium parvum,

On D10 post-immunostimulation, the mouse pups in batch 1 and 2 weresacrificed to evaluate protection against Cryptosporidium parvum.

1.5-Investigation of the State of Infection of the Mouse Pups

The state of infection of the mouse pups was analysed by the techniqueof tissue dissemination of the tachyzoites on HFF cells described above.The HFF cells are deposited in a 24-well plate, one week beforedepositing the organs at a rate of 10⁴ cells per well. The cells arecultured in 1 mL of DMEM cell culture medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin. After sacrifice of the mouse pups, the spleen ofeach of the mouse pups is taken and ground in 2 mL of 1×PBS. Tenmicrolitres of ground material is deposited per well containing the HFFcells cultured in 1 mL of medium. Twenty-four hours after depositing theground material from the spleen, the cells are washed in DMEM and then 1mL of clean medium is deposited per well. The cells are cultured untilthe lysis plaques revealing the presence of Toxo mic1-3 KO tachyzoitesare detected.

1.6-Investigation of Protection

The protection of the mouse pups against Cryptosporidium parvum isanalysed by counting the oocysts in the intestine. Once the mouse pupshave been sacrificed, the intestines are recovered, weighed, placed in 1mL of water (4° C.) and ground for 20 seconds. 100 microlitres of groundmaterial is added to 400 μl of sugar solution at 4° C. (500 g ofsucrose, 320 mL of distilled water, Na azide at 0.02%). Afterhomogenizing the solution by pipetting, 20 μL is deposited on a Thomaslide. Before counting the oocysts, the slides are kept in a cool placefor 15 minutes to allow the oocysts in the sugar solution to rise to thesurface. The total number of oocysts in the intestine is calculated fromthe following formula: Total number of oocysts of C. parvum in theintestine=Number of oocysts counted on Thoma slide×dilutionfactor×10,000×(1+weight of the intestine of the mouse pup).

2-Results

2.1-Investigation of the State of Infection of the Mouse Pups

Dissemination of the Spleens on HFF Cells

Dissemination of the spleens on MT′ cells is illustrated in Table XI.

TABLE XI Summary table of parasitaemia observed in the case of C. parvumand in the case of T. gondii of the batch of the mouse pups infectedwith C. parvum (infected C. parvum) and of the batch of the mouse pupsinoculated with Toxo mic1-3 KO and infected with C. parvum (inoculatedT. gondii + C. parvum). Inoculated C. parvum Inoculated T. gondii + Cparvum Total Total number of Parasit- number of Parasit- mouse oocystsof aemia mouse oocysts of aemia pups C. parvum T. gondii pups C. parvumT. gondii 1 2240000 NO 1 260000 YES 2 1794000 NO 2 476000 YES 3 1370000NO 3 2046000 SLIGHT 4 2448000 NO 4 1687500 SLIGHT

The mouse pups infected only with C. parvum have a total number ofoocysts of C. parvum in the intestine in the range from 1.4×10⁶ to2.4×10⁶ oocysts.

The mouse pups immunostimulated with Toxo mic1-3 KO and then infectedwith C. parvum display T. gondii parasitaemia determinedsemiquantitatively. For the mouse pups No. 1 and 2, which have severe T.gondii parasitaemia, the total number of oocysts of C. parvum in theintestine varies from 2.6×10⁵ to 4.8×10⁵ oocysts. These resultsdemonstrate the correlation that exists between parasitaemia with T.gondii mic1-3 KO and the reduction in infection with C. parvum.

2.2-Investigation of Protection

The state of protection of the mouse pups is shown in FIG. 12.

In the batch of the mouse pups vaccinated with the mutant Toxo mic1-3 KOby oral route, two mouse pups out of 4 have a total number of oocysts ofC. parvum in the intestine that is significantly reduced byapproximately 75% and 87%. These are the mouse pups No. 1 and No. 2.

Example 7 Induction of Secretion of IL-12 and of IFN-γ of Splenocytesand Induction of Secretion of IL-12 of MLN Cells Originating from Adultor Neonate Sheep after Stimulation with the Mutant Toxo Mic1-3 KO1-Experimental Protocol

1.1-Animals

The mesenteric lymph nodes and the spleens of neonates used in thisexperiment originate from lambs of the Ile de France breed aged 6-12days. Until sacrifice, the lambs were kept with their mothers in asealed sheep house (INRA-Nouzilly) in order to limit the risks ofnatural contamination. They were anaesthetized by electronarcosis andthen euthanased to collect the different organs. Only the animal keepersand the experimenters, equipped with clothing for use inside the sheephouse, may enter the buildings, in order to avoid contamination of theenvironment, and they only leave the sealed zone after showering. Theutensils used and the biological material are only taken out afterpassing through a disinfectant bath, and the waste is incinerated.

The mesenteric lymph nodes and the spleens of adult subjects used inthis experiment originate from adult sheep of the Ile de France breedaged from 1 to 3 years.

1.2-Strains of Toxoplasma gondii

The wild-type RH and Pru strains and the mutant strain Toxo mic1-3 KOare maintained by successive passages on a human foreskin fibroblast(HFF) line cultured in DMEM medium supplemented with 10% of foetal calfserum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 μg/mL ofstreptomycin.

1.3-Isolation of the Cells of Interest

1.3.1-Cells of the Mesenteric Lymph Nodes

The mesenteric lymph nodes are removed as quickly as possible after theanimals are euthanased, and transferred to a sterile sample tubecontaining culture medium (HBSS supplemented with 2% foetal calf serum(FCS) and 1% penicillin/streptomycin (P/S)). The sample tube containingthe sample is stored in ice until it reaches the research laboratory.

Each lymph node is defatted using previously sterilized forceps,scissors or scalpel. The lymph node is then deposited on a sterile(autoclaved) nylon mesh measuring 3 cm×3 cm placed at the bottom of asterile Petri dish containing 10 mL of medium (HBSS; 2% FCS; 1% P/S).The lymph node is crushed and comminuted using a piston of a 5-mLsyringe to release the cells contained in the lymph node. The mediumthus enriched with cells is filtered using a 60-μm sterile nylon clothpositioned above a 50-mL tube for recovering the medium enriched withfiltered cells. These last steps are repeated twice as follows: 10 mL ofmedium is added to the remaining fragments of lymph nodes. The fragmentsof lymph nodes are crushed, comminuted and filtered as described above.The 50-mL tube for recovering the filtered cell-enriched medium iscentrifuged at 1600 g for 15 min at 4° C. Washing is carried out with 50mL of medium (HBSS; 2% FCS; 1% P/S) by centrifugation for 10 min at 400g at 4° C. The pellet thus obtained is resuspended in a final volume of90 mL of medium (HBSS; 2% FCS; 1% P/S) at ambient temperature in two50-mL tubes.

The 90 mL of cell suspension is divided into 3 to be purified in aFicoll-Hypaque gradient. 30 mL is carefully deposited per tube (3 tubesin total) containing 15 mL of Ficoll-Hypaque. The 3 tubes ofFicoll-Hypaque enriched with cell suspension are centrifuged at 1500 gwithout braking (deceleration 2, acceleration 5), for 30 minutes at roomtemperature. For each tube of Ficoll-Hypaque, an upper phase is obtainedcomposed of cell debris, a ring composed of mononuclear cells,positioned as an interphase between the cell debris and the Ficollphase, and finally a lower phase composed of red blood cells. The 3rings (20 mL/tubes) are recovered and divided into two 50-mL tubes andwashed with a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S) at700 g for 10 min. The pellets of mononuclear cells obtained are pooled.The final pellet is washed with 50 mL of medium (HBSS; 2% FCS; 1% P/S)by centrifugation at 400 g for 10 min. The pellet is taken up in 5 mL ofRPMI, 10% FCS, 1% P/S, 5.10⁻⁵M of beta-mercaptoethanol. The mononuclearcells are stored in ice; a proportion of the cells is used for countingand observation of viability with Trypan blue (sample diluted to 1/100).Once counted, the cells are distributed in a 96-well plate at a rate of3.10⁵ cells/well.

1.3.2-Isolation of the Splenocytes

The spleen is removed as quickly as possible after euthanasia andtransferred to a sterile sample tube containing culture medium (HBSS; 2%FCS; 1% P/S). The sample tube containing the sample is stored in iceuntil it reaches the research laboratory.

Each spleen is defatted using previously sterilized forceps, scissors orscalpel. The spleen is then deposited on a metal grid (autoclaved)placed at the bottom of a sterile Petri dish containing 10 mL of medium(HBSS; 2% FCS; 1% P/S). The spleen is crushed and comminuted using apiston of a 5-mL syringe to release the cells contained in the spleen.The medium thus enriched with cells is deposited in a 50-mL tube. Theselast steps are repeated twice as follows: 5 mL of medium is added to thefragments of spleen. The fragments of spleen are crushed and comminutedas described above. The 50-mL tube for recovery of the medium enrichedwith cells is left to settle for 5 min at 4° C. The supernatant isfiltered on a 60 μm nylon mesh above a 50-mL tube. Ten millilitres ofmedium is added to the remaining sediments and the second supernatant isrecovered in the same way. The 50-mL tube for recovery of the twosupernatants is centrifuged for 30 s at 400 g at 4° C. The supernatantis recovered and filtered on a 60-μm sterile nylon mesh above a 50-mLtube. This last tube is centrifuged for 10 min at 400 g at 4° C. Thecell pellet thus obtained is resuspended in a final volume of 50 mL ofmedium (HBSS; 2% FCS; 1% P/S). Washing is carried out by centrifugationfor 10 min at 400 g at 4° C. The pellet is then taken up in a finalvolume of 120 mL in three 50-mL tubes of medium (HBSS; 2% FCS; 1% P/S)at room temperature.

The 120 mL of cell suspension is divided into 3 to be purified in aFicoll-Hypaque gradient. 30 mL is carefully deposited per tube (4 tubesin total) containing 15 mL of Ficoll-Hypaque. The 4 tubes ofFicoll-Hypaque enriched with cell suspension are centrifuged at 1500 gwithout braking (deceleration 2, acceleration 5), for 30 minutes at roomtemperature. For each tube of Ficoll-Hypaque, an upper phase is obtainedcomposed of cell debris, a ring composed of mononuclear cells positionedas an interphase between the cell debris and the Ficoll phase, andfinally a lower phase composed of red blood cells. The 4 rings (20mL/tubes) are recovered and divided into three 50-mL tubes and washedwith a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S) at 700 gfor 10 min. The pellets of mononuclear cells obtained are pooled. Thefinal pellet is washed with 50 mL of medium (HBSS; 2% FCS; 1% P/S) bycentrifugation at 400 g for 10 min. The pellet is taken up in 5 mL ofRPMI, 10% FCS, 1% P/S, 5.10⁻⁵M of beta-mercaptoethanol. The mononuclearcells are stored in ice; a proportion of the cells is used for countingand observation of viability with trypan blue (sample diluted to 1/100).Once counted, the cells are distributed in a 96-well plate at a rate of3.10⁵ cells/well.

1.4-Stimulation of the Mononuclear Cells of the Lamb Mesenteric LymphNodes and Splenocytes

Once counted, the cells are distributed in a 96-well plate at a rate of3.10⁵ cells/well. The tachyzoites are also counted on a Malassey slide;pellets are obtained and are taken up in RPMI medium, 10% FCS, 1% P/S.The solutions of the tachyzoites (RH or KO) are adjusted to obtain aconcentration of 3.10⁶ tachyzoites/mL. The conditions for stimulationare as follows: 3 mononuclear cells are stimulated per tachyzoite.Parasites, lymph node mononuclear cells and spleen mononuclear cells arecultured at 37° C. in a stove at 5% CO₂ and 95% humidity.

24 h post-stimulation, the supernatants of the lymph node mononuclearcells and the supernatants of the spleen mononuclear cells are used forassay of interleukin-12 and IFN-γ by the ELISA technique.

1.5-Assay of Interleukin-12 by the ELISA Technique

The technique used for assay of interleukin-12 is an ELISA of thesandwich type on a 96-well plate. The antibody (anti-IL-12) immobilizedon a plate reacts specifically with the IL-12 present in the testsample. The quantity of antigen-antibody is measured for a second timeafter it has reacted with an antibody of identical specificity coupledto an enzyme.

All the dilutions of the different reagents are carried out with 1×PBS,0.05% Tween 20 and 1% BSA except for the capture antibody, which isdiluted in 1×PBS.

50 μL per well of capture antibodies diluted to 1/500 (Mouse anti-bovineinterleukin-12 clone CC 301, Serotec MCA1782EL, Initial concentration:1000 μg/mL) is deposited in an ELISA plate (ELISA plate Nunc maxisorp442404) and then incubated overnight at 4° C. Three washings are carriedout in 1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T). Thenon-specific sites are saturated by incubating the plates for 1 hour atroom temperature with 200 μL of a 1×PBS solution; 0.05% Tween 20, 1%BSA. Three washings are carried out in 1×PBS buffer with the addition of0.05% Tween 20 (PBS-T).

The range is prepared as follows: 50 μL of the standard range of rovIL-12 is deposited at different concentrations: 16 U/mL, 8 U/mL, 4 U/mL,2 U/mL, 1 U/mL, 0.5 U/mL, 0.25 U/mL, 0.125 U/mL, 0.0625 U/mL, 0.03125U/mL. In parallel, the samples (supernatant of stimulated mononuclearcells, diluted 1/2) are deposited at a rate of 50 μL per well. Thesamples and the standard range are incubated for 1 h at roomtemperature. 4 washings are carried out in 1×PBS buffer with theaddition of 0.05% Tween 20 (PBS-T).

50 μL of biotinylated detection antibodies (Mouse anti-bovineinterleukin-12: biotin clone CC 326 (Serotec MCA2173B) Initialconcentration: 500 μg/ml) diluted to 1/500 is deposited per well, for anincubation time of one hour at room temperature. A series of 4 washingsis carried out in 1×PBS buffer with the addition of 0.05% Tween 20(PBS-T).

IL-12 is detected by a biotin-ExtrAvidine® affinity reaction followed bya colorimetric reaction between peroxidase and its substrate: 50 μL ofmodified avidin coupled to a peroxidase (ExtrAvidine-Peroxidaseconjugate® (Sigma E2886)) diluted to 1/2000th is deposited per well andincubated for 20 minutes at room temperature. A series of 4 washings iscarried out in 1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T).Detection is carried out using 50 μL/well of peroxidase substrateoriginating from a solution A and B mixed volume for volume (TMBPeroxidase substrate Eurobio KPL solution A 50-76-01 and solution B50-65-00). The substrate solution is incubated for 15 minutes at roomtemperature. 50 μL of stopping solution (phosphoric acid 1M Sigma43,080-1, CAS 7664-38-2) is added per well under a fume hood.

The plates are read at wavelength 2=450 nm. The standard curve isdetermined by the following function: OD value as a function of theconcentration of recombinant IL-12. The OD values of the samples testedare converted to concentration using the standard curve. To be takeninto account, the samples must give ODs located on the linear portion ofthe standard curve.

1.6-Assay of Interferon Gamma by the ELISA Technique

The technique used for assay of interferon is an ELISA, comparable tothe assay of interleukin-12 described in the preceding paragraph, of thesandwich type on a 96-well plate. The antibody (anti-IFNγ) immobilizedon a plate reacts specifically with the IFNγ present in the test sample.The quantity of antigen-antibody is measured for a second time after ithas reacted with an antibody of identical specificity to an enzyme.

All the dilutions of the different reagents are prepared in 1×PBS, 0.05%Tween 20 and 1% BSA except for the capture antibody, which is diluted in1×PBS.

50 μl per well of capture antibodies diluted to 1/500 (Mouse anti-bovineinterferon gamma clone CC 330, Serotec MCA2112, Initial concentration:1000 μg/mL) is deposited in an ELISA plate (ELISA plate Nunc maxisorp442404) and then incubated overnight at 4° C. Three washings are carriedout in 1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T). Thenon-specific sites are saturated by incubating the plates for 1 hour atroom temperature with 200 μL of a solution 1×PBS; 0.05% Tween 20, 1%BSA. Three washings are carried out in 1×PBS buffer with the addition of0.05% Tween 20 (PBS-T).

The range is prepared as follows: 50 μL of the standard range ofRecombinant bovine IFNγ (Perbio Endogen robIFNGγ, initial concentration:30 μg/ml) is deposited at different concentrations: 4 ng/mL, 2 ng/mL, 1ng/mL, 0.5 ng/mL, 0.25 ng/mL, 0.125 ng/mL, 0.0625 ng/mL, 0.03125 ng/mL,0.015 ng/ml). In parallel, the samples (supernatant of stimulatedmononuclear cells, diluted 1/2) are deposited at a rate of 50 μL perwell. The samples and the standard range are incubated for 1 h at roomtemperature. 4 washings are carried out in 1×PBS buffer with theaddition of 0.05% Tween 20 (PBS-T).

50 μL of biotinylated detection antibodies (Mouse anti-bovine interferongamma: biotin clone CC 302 (Serotec MCA1783B) Initial concentration: 500μg/ml) diluted to 1/500 is deposited per well, for an incubation time ofone hour at room temperature. A series of 4 washings is carried out in1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T).

IFNγ is detected by a biotin-ExtrAvidine® affinity reaction followed bya colorimetric reaction between peroxidase and its substrate: 50 μL ofmodified avidin coupled to a peroxidase (ExtrAvidine-Peroxidaseconjugate® (Sigma E2886)) diluted to 1/2000th is deposited per well andincubated for 20 minutes at room temperature. A series of 4 washings iscarried out in 1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T).Detection is carried out using 50 μL per well of peroxidase substrateoriginating from a solution A and B mixed volume for volume (TMBPeroxidase substrate Eurobio KPL solution A 50-76-01 and solution B50-65-00). The substrate solution is incubated for 15 minutes at roomtemperature. 50 μL of stopping solution (phosphoric acid 1M Sigma43,080-1, CAS 7664-38-2) is added per well under a fume hood.

The plates are read at wavelength 2=450 nm. The standard curve isdetermined by the following function: OD value as a function of theconcentration of recombinant IFNγ. The OD values of the samples testedare converted to concentration using the standard curve. To be takeninto account, the samples must give ODs located on the linear portion ofthe standard curve.

2-Results

The level of IL-12 and of IFN-γ secreted by the mononuclear cells ofspleens and the level of IL-12 secreted by the mononuclear cells ofmesenteric lymph nodes (MLN) isolated from lambs aged from 6 to 12 daysand from adult sheep aged from 1 to 3 years and stimulated withdifferent strains of the parasite Toxoplasma gondii are shown in FIGS.13-A, 13-B and 13-C.

In the sheep aged from 1 to 3 years, production of IL-12 by themononuclear cells originating from the spleen or from the mesentericlymph nodes is greater when the latter have been stimulated in vitro bythe mutant strain Toxo mic1-3 KO or by the wild-type Pru and RH strainsof T. gondii. This observation is more pronounced in the case of cellsfrom the mesenteric lymph nodes of adult sheep.

In the lambs aged from 6 to 12 days, production of IL-12 by themononuclear cells originating from the spleen or from the mesentericlymph nodes is greater when the latter have been stimulated in vitro bythe parasite T. gondii. Production of IL-12 is even greater when themononuclear cells have been stimulated in vitro by the mutant strainToxo mic1-3 KO.

In the sheep aged from 1 to 3 years, production of IFNγ by themononuclear cells originating from the spleen does not differsignificantly when the latter have been stimulated in vitro by themutant strain mic1-3 KO or by the wild-type strains Pru and RH of T.gondii.

In the lambs aged from 6 to 12 days, production of IFNγ by themononuclear cells originating from the spleen does not differsignificantly when the latter have been stimulated in vitro by themutant strain mic1-3 KO or by the wild-type strains Pru and RH of T.gondii.

Production of IL-12 and IFN-γ by the mononuclear cells originating fromlambs is much greater than that observed for the mononuclear cells fromthe adult sheep.

Example 8 Immunostimulation of Neonate Lambs by the Mutant Toxoplasmagondii Toxo Mic1-3 KO 1-Experimental Protocol

1.1-Animals

Immunostimulation is carried out on 1-day-old neonate lambs. Afteringestion of colostrum, the lambs were isolated from their mothers in asealed sheep house (INRA-Nouzilly) in order to limit the risks ofnatural contamination. They were anaesthetized by electronarcosis andthen euthanased to collect the various organs. Only the animal keepersand the experimenters, equipped with clothing for use inside the animalhouse, may enter the buildings, to prevent contamination of theenvironment, and they only leave the sealed zone after showering.

1.2-Strain Toxo mic1-3 KO

The mutant strain of Toxoplasma gondii, in which the genes coding forthe proteins MIC1 and MIC3 have been suppressed (called strain Toxomic1-3 KO) is maintained by successive passages on a human foreskinfibroblast (HFF) line cultured in DMEM medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin.

1.3-Immunostimulation

Six one-day-old lambs were treated as follows:

4 lambs (batch A) received the mutant Toxo mic1-3 KO

2 lambs (batch B) served as unvaccinated control batch.

On D1, the lambs in batch A received 10⁶ tachyzoites of the strain Toxomic1-3 KO subcutaneously. After immunostimulation, the temperatures andthe weights of the lambs are recorded daily.

On D15 post-immunostimulation, the lambs are euthanased forinvestigating the immune response and parasitaemia.

1.4-Isolation of the Cells of Interest

1.4.1-Isolation of the Splenocytes

The spleen is removed as quickly as possible after euthanasia andtransferred to a sterile sample tube containing culture medium (HBSS; 2%FCS; 1% P/S). The sample tube containing the sample is stored in iceuntil it reaches the research laboratory.

Each spleen is defatted using previously sterilized forceps, scissors orscalpel. The spleen is then deposited on a metal grid (autoclaved)placed at the bottom of a sterile Petri dish containing 10 mL of medium(HBSS; 2% FCS; 1% P/S). The spleen is crushed and comminuted using apiston of a 5-mL syringe to release the cells contained in the spleen.The medium thus enriched with cells is deposited in a 50-mL tube. Theselast steps are repeated twice as follows: 5 mL of medium is added to thefragments of spleen. The fragments of spleen are crushed and comminutedas described above. The 50-mL tube for recovery of the cell-enrichedmedium is left to settle for 5 min at 4° C. The supernatant is filteredon a 60 μm nylon mesh above a 50-mL tube. Ten millilitres of medium isadded to the remaining sediments and the second supernatant is recoveredin the same way. The 50-mL tube for recovery of the two supernatants iscentrifuged for 30 s at 400 g at 4° C. The supernatant is recovered andfiltered on a 60-μm sterile nylon mesh above a 50-mL tube. This lasttube is centrifuged for 10 min at 400 g at 4° C. The cell pellet thusobtained is resuspended in a final volume of 50 mL of medium (HBSS; 2%FCS; 1% P/S). Washing is carried out by centrifugation for 10 min at 400g at 4° C. The pellet is then taken up in a final volume of 120 mL inthree 50-mL tubes of medium (HBSS; 2% FCS; 1% P/S) at room temperature.

The 120 mL of cell suspension is divided into 3 to be purified in aFicoll-Hypaque gradient. 30 mL is carefully deposited per tube (4 tubesin total) containing 15 mL of Ficoll-Hypaque. The 4 tubes ofFicoll-Hypaque enriched with cell suspension are centrifuged at 1500 gwithout braking (deceleration 2, acceleration 5), for 30 minutes at roomtemperature. For each tube of Ficoll-Hypaque, an upper phase is obtainedcomposed of cell debris, a ring composed of mononuclear cells positionedas an interphase between the cell debris and the Ficoll phase, andfinally a lower phase composed of red blood cells. The 4 rings (20mL/tubes) are recovered and divided into three 50-mL tubes and washedwith a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S) at 700 gfor 10 min. The pellets of mononuclear cells obtained are pooled. Thefinal pellet is washed with 50 mL of medium (HBSS; 2% FCS; 1% P/S) bycentrifugation at 400 g for 10 min. The pellet is taken up in 5 mL ofRPMI, 10% FCS, 1% P/S, 5.10⁻⁵M of beta-mercaptoethanol. The mononuclearcells are stored in ice; a proportion of the cells is used for countingand observation of viability with trypan blue (sample diluted to 1/100).Once counted, the cells are distributed in a 96-well plate at a rate of3.10⁵ cells/well.

1.4.2-Isolation of the Lymph Node Cells

The popliteal and subiliac lymph nodes, situated near the injectionsite, are removed as quickly as possible after euthanasia of the animalsand are transferred to a sterile sample tube containing culture medium(HBSS supplemented with 2% foetal calf serum (FCS) and 1%penicillin/streptomycin (P/S)). The sample tube containing the sample isstored in ice until it reaches the research laboratory.

Each lymph node is defatted using previously sterilized forceps,scissors or scalpel. The lymph node is then deposited on a sterile(autoclaved) nylon mesh measuring 3 cm×3 cm placed at the bottom of asterile Petri dish containing 10 ml of medium (HBSS; 2% FCS; 1% P/S).The lymph node is crushed and comminuted using a piston of a 5-mLsyringe to release the cells contained in the lymph node. The mediumthus enriched with cells is filtered using a 60-μm sterile nylon meshpositioned above a 50-mL tube for recovering the medium enriched withfiltered cells. These last steps are repeated twice as follows: 10 mL ofmedium is added to the remaining fragments of lymph nodes. The fragmentsof lymph nodes are crushed, comminuted and filtered as described above.The 50-mL tube for recovering the filtered cell-enriched medium iscentrifuged at 1600 g for 15 min at 4° C. Washing is carried out with 50mL of medium (HBSS; 2% FCS; 1% P/S) by centrifugation for 10 min at 400g at 4° C. The pellet is taken up in 5 mL of RPMI, 10% FCS, 1% P/S,5.10⁻⁵M of beta-mercaptoethanol. The pellet is stored in ice; aproportion of the cells is used for counting and observation ofviability with trypan blue (sample diluted to 1/100). Once counted, thecells are distributed in a 96-well plate at a rate of 3.10⁵ cells/well.

1.5-Investigation of the Inflammatory Response Post-Immunostimulation

1.5.1-from the Spleen Cells

Once the mononuclear cells of the spleen have been isolated by FicollHistopaque gradient and distributed in a 96-well plate, at a rate of3.10⁵ cells per well, the latter are stimulated in vitro with 30 μg/mlof total parasite extract of the strain Toxo mic1-3 KO. To establish apositive control for the experiments, the cells are stimulated withconcanavalin A. Conversely, to establish a negative control for theexperiments, the cells are cultured in a medium without stimulant. Thesupernatants are taken after 24 hours post-stimulation in vitro.

The technique used for assay of IFNγ is an ELISA of the sandwich type ona 96-well plate. The antibody (anti-IFNγ) immobilized on a plate reactsspecifically with the IFNγ present in the test sample. The quantity ofantigen-antibody is measured in a second phase after it has reacted withan antibody of identical specificity coupled to an enzyme.

All the dilutions of the various reagents are prepared in 1×PBS, 0.05%Tween 20 and 1% BSA, except for the capture antibody, which is dilutedin 1×PBS.

50 μL per well of capture antibody diluted to 1/500 (Mouse anti-bovineIFNg clone CC 330, Serotec MCA2112, Initial concentration: 1000 μg/mL)is deposited in an ELISA plate (ELISA plate Nunc maxisorp 442404) andthen incubated overnight at 4° C. Three washings are carried out in1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T). Thenon-specific sites are saturated by incubating the plates for 1 hour atroom temperature with 200 μL of a 1×PBS solution; 0.05% Tween 20, 1%BSA. Three washings are carried out in 1×PBS buffer with the addition of0.05% Tween 20 (PBS-T).

The range is prepared as follows: 50 μL of the standard range of rBoIFNγis deposited at different concentrations: 4 ng/mL, 2 ng/mL, 1 ng/mL, 0.5ng/mL, 0.25 ng/mL, 0.125 ng/mL, 0.0625 ng/mL, 0.03125 ng/mL, 0.015ng/mL. In parallel, the samples (supernatant of stimulated mononuclearcells) are deposited at a rate of 50 μL per well. The samples and thestandard range are incubated for 1 h at room temperature. 4 washings arecarried out in 1×PBS buffer with the addition of 0.05% Tween 20 (PBS-T).

50 μL of biotinylated detection antibodies (Mouse anti-bovine IFNg:biotin clone CC 302 (Serotec MCA1783B) Initial concentration: 500 μg/ml)diluted to 1/500 is deposited per well, for an incubation time of onehour at room temperature. A series of 4 washings is carried out in 1×PBSbuffer with the addition of 0.05% Tween 20 (PBS-T).

IFNγ is detected by a biotin-ExtrAvidine® affinity reaction followed bya colorimetric reaction between peroxidase and its substrate: 50 μL of asolution of modified avidin coupled to a peroxidase(ExtrAvidine-Peroxidase conjugate® (Sigma E2886)) diluted to 1/2000th isdeposited per well and incubated for 20 minutes at room temperature. Aseries of 4 washings is carried out in 1×PBS buffer with the addition of0.05% Tween 20 (PBS-T). Detection is carried out using 50 μL/well ofperoxidase substrate originating from a solution A and B mixed volumefor volume (TMB Peroxidase substrate Eurobio KPL solution A 50-76-01 andsolution B 50-65-00). The substrate solution is incubated for 15 minutesat room temperature. 50 μL of stopping solution (phosphoric acid 1MSigma 43,080-1, CAS 7664-38-2) is added per well under a fume hood.

The plates are read at wavelength λ=450 nm. The standard curve isdetermined by the following function: OD value as a function of theconcentration of the recombinant IFNγ. The OD values of the samplestested are converted to concentration using the standard curve. To betaken into account, the samples must give ODs located on the linearportion of the standard curve.

1.5.2-from the Lymph Node Cells

The lymph node cells distributed in a 96-well plate at a rate of 3.10⁵cells per well are cultured without any stimulant in order to detectexpression of the cytokines ex-vivo. The supernatants of the lymph nodecells are taken after 24 hours. IFNγ is assayed by the ELISA techniqueas described above.

2-Results

2.1-Experimental Procedure

After ingestion of colostrum, the lambs are separated from their mothersand are fed with a lamb feeder bucket. The 4 lambs in batch A wereimmunostimulated with 10⁶ tachyzoites of the strain Toxo mic1-3 KO,freshly produced. After inoculation with the strain Toxo mic1-3 KO, nosevere clinical sign was found and the lambs immunostimulated with thestrain Toxo mic1-3 KO have a weight curve similar to the weight gain ofthe control lambs (FIG. 14).

The lambs from batch A and from batch B were euthanased 15 days afterimmunostimulation.

2.2-Investigation of the Inflammatory Response Post-Immunostimulation

The results of the assays of the IFN-γ produced from splenocytesrestimulated with total extract of T. gondii and from the cells of thesubiliac and popliteal lymph nodes are presented in FIGS. 15 and 16.

The lambs of the control batch (1423 and 1428) did not develop aninflammatory response (IFN-γ secretion), either in the spleen, or in thesubiliac and popliteal lymph nodes.

In contrast, 15 days after immunostimulation, the cells of the subiliaclymph nodes of 3 lambs out of 4 produce IFN-γ. These lymph nodes aresituated downstream of the injection site, in contrast to the popliteallymph nodes, situated upstream and in which no production of IFN-γ isdetected.

The inflammatory response of the lambs immunostimulated with the strainToxo mic1-3 KO is confirmed after restimulation of the splenocytes withtotal parasite extract of the strain Toxo mic1-3 KO, since production ofIFN-γ is induced for the 4 lambs. The splenocytes are also stimulatedwith concanavalin A, a protein of the lectin family, known to be apolyclonal activator of the T lymphocytes, which serves as positivecontrol of stimulation of the immune system cells.

Example 9 Protection Against Cryptosporidiosis of Lambs ImmunostimulatedSubcutaneously with the Mutant Toxo Mic1-3 KO 1-Experimental Protocol

1.1-Animals

Immunostimulation is carried out on 1-day-old neonate lambs. Afteringestion of colostrum, the lambs were isolated from their mothers in asealed sheep house (INRA-Nouzilly) in order to limit the risks ofnatural contamination.

At the end of the experiments, the lambs were anaesthetized byelectronarcosis and then euthanased to collect the various organs. Onlythe animal keepers and the experimenters, equipped with clothing for useinside the animal house, may enter the buildings, in order to preventcontamination of the environment, and they only leave the sealed zoneafter showering.

1.2-Cryptosporidium parvum

Oocysts of Cryptosporidium parvum are obtained from excrement of calvesinfected with 10⁷ C. parvum oocysts. The stool undergoes varioustreatments until a suspension of sterile, purified parasites isobtained, suitable for use in cell culture. Throughout the treatment,the oocysts are manipulated at 4° C. to prevent excystation of theoocysts.

Briefly, after recovery of the stool, the latter is diluted in freshwater and then passed through a 100-μm filter and centrifuged at 1900 gfor 10 minutes at 4° C. The pellets obtained, containing the oocysts,are taken up in 2% potassium dichromate solution (Prolabo, ref. 26 776290, CAS 7778-50-9), and are then washed twice with cold water bycentrifugation at 1900 g for 10 minutes at 4° C. to remove the potassiumdichromate. After washing, the pellet of coccidia is taken up in amixture of water and ether (ethyl ether, Carlo Erba, CAS No. 60-29-7)diluted to 1/5, then centrifuged again at 1900 g for 10 minutes at 4° C.The upper phases containing the fats and the ether are removed and thepellet is recovered and taken up in cold water after passing through a20-μm filter. Two to three millilitres of the suspension of parasitesobtained is deposited on a glucose gradient prepared from Sheathersolution (sucrose 500 g, water 320 ml, 0.2 g of sodium azide (Prolabo,CAS 26628-22-8)). Two rings of oocysts are formed after centrifugationof the glucose gradient at 2000 g for 20 minutes at 4° C. The two ringsare recovered and washed several times in cold water. The purifiedoocysts are then sterilized. After centrifugation at 1900 g for 10minutes at 4° C., the pellet of oocysts is incubated for 15 minutes in asolution of bleach (sodium hypochlorite (Sigma 239305-500ML Titre: 4.5%of active chlorine)) diluted to 10% in demineralized water, then washed3 times in sterile 1×PBS (diluted from a solution of PBS 10×: sodiumchloride, NaCl 80 g/litre water; potassium chloride, KCl 2 g/litre;potassium dihydrogen phosphate, KH₂PO₄: 2 g/litre; disodium hydrogenphosphate, Na₂HPO₄, 12 H₂O: 29 g/litre). The purified and sterilizedoocysts are counted on a slide (5 μL of solution of oocysts and 495 μLof malachite green), adjusted to a concentration of 2×10⁸ oocysts/mL,divided into aliquots in 1.5-mL tubes and stored at 4° C.

1.3-Strain Toxo Mic1-3 KO

The mutant strain of Toxoplasma gondii, in which the genes coding forthe proteins TgMIC1 and TgMIC3 have been knocked out (called strain Toxomic1-3 KO) is maintained by successive passages on a human foreskinfibroblast (HFF) line cultured in DMEM medium supplemented with 10% offoetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50μg/mL of streptomycin.

1.4-Immunostimulation

Two batches of 1-day-old male lambs were treated as follows:

8 lambs (batch A) served as unvaccinated control batch,

-   -   8 lambs (batch B) received the mutant Toxo mic1-3 KO.

On D1, the lambs in batch B received 10⁶ tachyzoites of the strain Toxomic1-3 KO by intraperitoneal route.

On D7 post-infection, the lambs in batch A and in batch B werechallenged with 5.10⁶ oocysts of Cryptosporidium parvum and the lambswere sacrificed on D31.

1.5-Investigation of Protection

To analyse the protection generated by immunostimulation with the strainToxo mic1-3 KO, 3 parameters were studied:

Survival-Mortality

After challenge with Cryptosporidium parvum, the lambs were monitoreddaily for 25 days (until D31).

Weight Gain:

The lambs in batch A and in batch B were weighed every 2-3 days toevaluate their weight gain.

Excretion of Oocysts:

The faeces of the lambs were recovered daily from D9 to D22 and werestored at +4° C. After weighing, 26 mg of faecal matter is diluted in750 μL of water. 4 mL of sucrose solution is added. Afterhomogenization, 20 μL is deposited on a Thoma slide and the oocysts arecounted. The number of oocysts excreted is then calculated with thefollowing formula: N=n×10 000×20 (N: number of oocysts per g or per ml,n=number of oocysts counted on the cell, 20=dilution 1/4×1/5).

2-Results

2.1-Experimental procedure

After ingestion of colostrum, the lambs are separated from their motherand are fed with a lamb feeder bucket. 3 lambs from batch B and 1 lambfrom batch A were unable to feed properly and were euthanased. Sinceeuthanasia took place before the infectious challenge, these animalswere statistically removed from the protocol.

2.1 Investigation of Protection

Survival-Mortality

Following infection with C. parvum, four lambs essentially of thecontrol batch (batch A) were euthanased, in contrast to theimmunostimulated batch (batch B), in which no lamb died followinginfection with C. parvum (FIG. 17).

Weight Gain

The daily weight gain (DAG, daily average gain) of the lambs in batch Aand in batch B is presented in FIG. 18. A significantly lower DAG isfound for batch A challenged only with C. parvum than for batch Bimmunostimulated with the strain Toxo mic1-3 KO and then challenged withC. parvum.

Excretion of Oocysts

Analysis of the parasitic load in the excrement of the lambs shows ashift of the excretion peak of one day for the batch immunostimulatedwith the strain Toxo mic1-3 KO, with a 70% drop of the mean excretionpeak observed at 5 days post-infection with C. parvum compared to themean excretion peak for the control batch challenged only with C. parvumobserved at 4 days post-infection with C. parvum (FIG. 19).

1. Strains of Sarcocystidae selected from Toxoplasma spp or Neospora sppisolated from their natural environment and having an immunostimulanteffect, for the use thereof in the prevention or the treatment, in aneonate mammal, of a pathology associated with an apicomplexan of thefamily Cryptosporidiidae.
 2. Strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, for the use thereof according toclaim 1, in which said mammal is a human being or an animal.
 3. Strainsof Sarcocystidae selected from Toxoplasma spp or Neospora spp isolatedfrom their natural environment and having an immunostimulant effect, forthe use thereof according to claim 2, in which said animal belongs tothe group comprising or constituted by ovines, caprins, porcines,bovines, equines, camelids, canids or felids.
 4. Strains ofSarcocystidae selected from Toxoplasma spp or Neospora spp isolated fromtheir natural environment and having an immunostimulant effect, for theuse thereof according to claim 1, in which said strains of Toxoplasmaspp or of Neospora spp have at least an adhesin MIC-1 and/or an adhesinMIC-3 inactivated by a genetic modification relating to at least one ofthe mic-1 and/or mic-3 genes.
 5. Strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, for the use thereof according toclaim 1, in which said strains of Toxoplasma spp or of Neospora spp havethe two adhesins MIC-1 and MIC-3 inactivated by a genetic modificationrelating to the two mic-1 and mic-3 genes.
 6. Strains of Sarcocystidaeselected from Toxoplasma spp or Neospora spp isolated from their naturalenvironment and having an immunostimulant effect, for the use thereofaccording to claim 4, in which the strains of Toxoplasma spp or ofNeospora spp are respectively Toxoplasma gondii or Neospora caninum. 7.Strains of Sarcocystidae selected from Toxoplasma spp or Neospora sppisolated from their natural environment and having an immunostimulanteffect, for the use thereof according to claim 1, in which theimmunostimulant effect of said strains leads to the secretion ofinterleukin-12 (IL-12) and then of interferon-γ (IFN-γ).
 8. Strains ofSarcocystidae selected from Toxoplasma spp or Neospora spp isolated fromtheir natural environment and having an immunostimulant effect, for theuse thereof according to claim 7, in which the secretion ofinterleukin-12 (IL-12) and of interferon-γ (IFN-γ) begins between 3 and9 days after using said strains of Toxoplasma spp or of Neospora spp asimmunostimulant.
 9. Strains of Sarcocystidae selected from Toxoplasmaspp or Neospora spp isolated from their natural environment and havingan immunostimulant effect, for the use thereof according to claim 1, inwhich said pathology associated with an apicomplexan of the familyCryptosporidiidae is cryptosporidiosis.
 10. Strains of Sarcocystidaeselected from Toxoplasma spp or Neospora spp isolated from their naturalenvironment and having an immunostimulant effect, for the use thereofaccording to claim 9, in which the apicomplexan of the familyCryptosporidiidae responsible for the cryptosporidiosis is at least oneapicomplexan selected from the group constituted by Cryptosporidiumparvum, Cryptosporidium bovis, Cryptosporidium andersoni,Cryptosporidium ryanae, Cryptosporidium muris, Cryptosporidiumubiquitum, Cryptosporidium hominis, Cryptosporidium canis,Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidiummeleagridis or Cryptosporidium xiaoi.
 11. Strains of Toxoplasma gondiior of Neospora caninum isolated from their natural environment andhaving an immunostimulant effect, said strains of Toxoplasma gondii orof Neospora caninum having the two adhesins MIC-1 and MIC-3 inactivatedby a genetic modification relating to the two mic-1 and mic-3 genes, forthe use thereof according to claim 6, in which said pathology associatedwith an apicomplexan of the family Cryptosporidiidae iscryptosporidiosis.
 12. Strains of Toxoplasma gondii or of Neosporacaninum isolated from their natural environment and having animmunostimulant effect, for the use thereof in the prevention or thetreatment, in a mammal, of a pathology associated with an apicomplexanof the family Cryptosporidiidae, said strains of Toxoplasma gondii or ofNeospora caninum having the two adhesins MIC-1 and MIC-3 inactivated bya genetic modification relating to the two mic-1 and mic-3 genes, andsaid pathology associated with an apicomplexan of the familyCryptosporidiidae is cryptosporidiosis.
 13. Strains of Toxoplasma gondiior of Neospora caninum isolated from their natural environment andhaving an immunostimulant effect, said strains of Toxoplasma gondii orof Neospora caninum having the two adhesins MIC-1 and MIC-3 inactivatedby a genetic modification relating to the two mic-1 and mic-3 genes, forthe use thereof according to claim 6, in which said strains ofToxoplasma gondii or of Neospora caninum are administered to the mammalat a rate from 20 to 10⁹ tachyzoites.
 14. Strains of Sarcocystidaeselected from Toxoplasma spp or Neospora spp isolated from their naturalenvironment and having an immunostimulant effect, for the use thereofaccording to claim 1, in which said strains are in a galenic formselected from the group comprising or constituted by liquid suspensions,solid or liquid dispersions, powders, pastes or lyophilizates. 15.Strains of Sarcocystidae selected from Toxoplasma spp or Neospora sppisolated from their natural environment and having an immunostimulanteffect, for the use thereof according to claim 1, in which said strainsare associated with at least one other antigen, or at least oneadjuvant, or at least one stabilizer, or at least one preservative or amixture of at least two of said products for increasing the immuneresponse of said mammal.
 16. Strains of Sarcocystidae selected fromToxoplasma spp or Neospora spp isolated from their natural environmentand having an immunostimulant effect, for the use thereof according toclaim 5, in which the strains of Toxoplasma spp or of Neospora spp arerespectively Toxoplasma gondii or Neospora caninum.